Rapamycin analogs and uses thereof

ABSTRACT

The present invention provides compounds, compositions thereof, and methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional App. No.62/685,666 filed on Jun. 15, 2018, the content of which is herebyincorporated by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to compounds and methods useful formodulating mTORC1 activity. The invention also provides pharmaceuticallyacceptable compositions comprising provided compounds of the presentinvention and methods of using such compositions in the treatment ofvarious disorders.

BACKGROUND OF THE INVENTION

mTOR complex 1 (mTORC1) positively regulates cell growth andproliferation by promoting many anabolic processes, includingbiosynthesis of proteins, lipids and organelles, and by limitingcatabolic processes such as autophagy. Much of the knowledge aboutmTORC1 function comes from the use of the bacterial macrolide rapamycin.Upon entering the cell, rapamycin binds to FK506-binding protein of 12kDa (FKBP12) and interacts with the FKBP12-rapamycin binding domain(FRB) of mTOR, thus inhibiting mTORC1 functions (Guertin, D. A. &Sabatini, D. M. Cancer Cell 12(1): 9-22 (2007)). In contrast to itseffect on mTORC1, FKBP12-rapamycin cannot physically interact with oracutely inhibit mTOR complex 2 (mTORC2) (Janinto, E. et al., Nat. CellBio., 6(11): 1122-8 (2004); Sarbassov, D. D. et al., Curr. Biol. 14(14):1296-302 (2004)). On the basis of these observations, mTORC1 and mTORC2have been respectively characterized as the rapamycin-sensitive andrapamycin-insensitive complexes. However, this paradigm might not beentirely accurate, as chronic rapamycin treatment can, in some cases,inhibit mTORC2 activity by blocking its assembly (Sarbassov, D. D. etal., Mol. Cell, 22(2): 159-68 (2006)). In addition, recent reportssuggest that important mTORC1 functions are resistant to inhibition byrapamycin (Choo, A. Y. et al., Proc. Natl. Acad. Sci., 105(45): 17414-9(2008); Feldman, M. E. et al., PLoS Biol., 7(2):e38 (2009);Garcia-Martinez, J. M. et al., Biochem J., 421(1): 29-42 (2009);Thoreen, C. C. et al., J. Biol. Chem., 284(12): 8023-32 (2009)).Therefore, selective inhibition of mTORC1 would enable the treatment ofdiseases that involve dysregulation of protein synthesis and cellularmetabolism. Furthermore, this detailed understanding of regulatingmTORC1 activation pathways will permit the discovery of new strategiesfor regulating abnormal disease processes by modulating mTORC1 activityacross its spectrum of function.

Many diseases are associated with abnormal cellular responses triggeredby events as described above. These diseases include, but are notlimited to, autoimmune diseases, inflammatory diseases, bone diseases,metabolic diseases, neurological and neurodegenerative diseases, cancer,cardiovascular diseases, allergies and asthma, Alzheimer's disease, andhormone-related diseases.

The mechanistic target of rapamycin complex 1 (mTORC1) is a mastergrowth regulator that senses diverse environmental cues, such as growthfactors, cellular stresses, and nutrient and energy levels. Whenactivated, mTORC1 phosphorylates substrates that potentiate anabolicprocesses, such as mRNA translation and lipid synthesis, and limitscatabolic ones, such as autophagy. mTORC1 dysregulation occurs in abroad spectrum of diseases, including diabetes, epilepsy,neurodegeneration, immune response, suppressed skeletal muscle growth,and cancer among others (Howell, J. J. et al., Biochem. Soc. Trans., 41:906-12 (2013); Kim, S. G. et al., Molecular and cells, 35(6): 463-73(2013); Laplante, M. & Sabatini, D. M., Cell, 149(2): 274-93 (2012)).

Rapamycin was initially discovered as an antifungal metabolite producedby Streptomyces hygroscopicus from a soil sample of Easter Island.Subsequently, rapamycin was found to possess immunosuppressive andantiproliferative properties in mammalian cells, spurring an interest inidentifying the mode of action of rapamycin. Rapamycin was shown to be apotent inhibitor of S6K1 phosphorylation. Concurrently, the target ofrapamycin (TOR) was identified in yeast and animal cells. Rapamycinforms a gain-of-function complex with the 12 kDa FK506-binding protein(FKBP12), and this complex binds and specifically acts as an allostericinhibitor of mammalian TOR (mTOR, also known as mechanistic TOR) complex1 (mTORC1).

Biochemical and genetic analysis of mTOR has demonstrated that it ispresent in two functionally distinct complexes. The core components ofmTORC1 consist of mTOR, mammalian lethal with sec-13 protein 8 (mLST8),and regulatory-associated protein of TOR (Raptor). Additional componentsinclude DEP-domain-containing mTOR-interacting protein (DEPTOR) andProline-rich Akt substrate 40 kDa (PRAS40).

The mTOR complex 2 (mTORC2) core is composed of mTOR, rapamycininsensitive companion of mTOR (Rictor), stress-activated proteinkinase-interacting protein 1 (mSIN1), and mLST8. Protein observed withrictor ½ (protor ½) and DEPTOR are additional regulatory components. S6kinase 1 (S6K1) and eukaryotic inhibition factor eIF4E binding protein 1(4E-BP1) are two well-characterized substrates of mTORC1 while AKT is awell characterized substrate of mTORC2 (Li, J. et al., Cell Met.,19(3):373-9 (2014)).

Because FKBP12-rapamycin does not bind to mTORC2, rapamycin wasinitially thoughtto inhibit only mTORC1 (Sarbassov, D. D. et al., Curr.Biol., 14(14):1296-302 (2004)). However, in 2006 it was shown thatrapamycin suppresses the assembly and function of mTORC2 and inhibitspAkt (Sarbassov, D. D. et al., Molecular Cell, 22(2): 159-68 (2006)).The effects of rapamycin on the phosphorylation of S473 of Akt (anmTORC2 substrate) and of T389 of S6K1 (an mTORC1 substrate) werecompared in multiple cell lines. In PC3, HEK-293T, HeLa, and H460 cells,1 or 24 hour treatments with rapamycin inhibited S6K1 phosphorylation,consistent with inhibition of mTORC1. Selective inhibition of S6K1 byrapamycin should lead to an increase in Akt phosphorylation, and,indeed, this is what is reported in HeLa cells. However, in PC3 cells,the drug strongly decreased Akt phosphorylation suggesting thatrapamycin is not selective in this cell line. Partial inhibition of pAKTis observed in HEK-293T cells. In about one third of the cell lines,rapamycin caused a strong or partial inhibition of Akt phosphorylation,while the drug either did not affect or increased Akt phosphorylation inthe others. The inhibition of pAKT after 24 hours is also observed inprimary and non-transformed cell lines including endothelial and musclecells. Rapamycin was also shown to inhibit pAkt in vivo, as mice treateddaily for 1 week with the drug had decreased Akt phosphorylation in thethymus, adipose tissue, heart, and lung. These findings demonstratedthat inhibition of Akt phosphorylation by rapamycin is common and occursin normal cell lines, cancer cell lines as well as in vivo.

It was concluded by Sarbassov et al. that rapamycin and its analogs (CCI779, RAD001 also known as Everolimus, AP23573) can inhibit mTORC2function in certain cell lines and tissues. Rapamycin-mediatedinhibition of Akt may help explain the side effects of the drug. Forexample, rapamycin strongly inhibits Akt phosphorylation in adiposetissue, a tissue type in which insulin-stimulated Akt activity plays animportant role in suppressing lipolysis. Inhibition of Akt by rapamycinin adipocytes may allow lipolysis to remain high even in the presence ofinsulin, resulting in the accumulation of free fatty acids in the plasmathat can be used by the liver to generate triglycerides, providing amolecular mechanism for the hyperlipidemia commonly seen in patientstreated with rapamycin.

Pereira et al. (Mol Cell Endocrinol., 355(1): 96-105 (2012)) exploredrapamycin effects on glucose uptake and insulin signaling proteins inadipocytes obtained via fat biopsies in human donors. At therapeuticconcentration (0.01 μM) rapamycin reduced AKT (PKB) Ser473phosphorylation and reduced glucose uptake in human adipocytes throughimpaired insulin signaling.

Lamming et al. (Science., 335(6076): 1638-1643 (2012)) demonstrated thatrapamycin disrupted mTORC2 in vivo and that mTORC2 was required for theinsulin-mediated suppression of hepatic gluconeogenesis.

Similar results were shown in human. Di Paolo et al. published similarfindings in human (JASN, 17(8): 2236-2244 (2006)). The main objective oftheir study was to ascertain the effect of chronic exposure to rapamycinon AKT activation, in view of its crucial role in the regulation of cellgrowth and survival, as well as in the cell response to nutrients andgrowth factors. They found that mTOR inhibition was associated with amarked downregulation of basal and insulin-induced AKT phosphorylation.AKT is responsible primarily for many of the metabolic actions ofinsulin and they concluded therefore that the depression of AKTactivation significantly correlated with the increase of insulinresistance in renal transplant recipients.

Kennedy et al. reviewed recently the role of mTORC1 and mTORC2 inmetabolism and aging (Cell Metab., 23(6): 990-1003 (2016)).

It has been surprisingly found that provided compounds inhibit mTORC1,but do not impact mTORC2 (as measured by their impact on pAKT) overextended periods of time (e.g., 8 hours, 24 hours, 30 hours, and 48hours). This novel activity is predicated on the presence of asufficiently large group at the C-7 position of rapamycin and itsanalogs. Small substitutions at this position such as OMe, as seen inrapamycin, OEt, OBn do not confer selectivity over mTORC2 at 24 hours.Medium length groups, such as OCH₂CH₂OH or OCH₂CH₂CH₂OH show partialselectivity over mTORC2 at 24 hours, but still show some level ofinhibition. In comparison, larger groups, such as those of the presentinvention (e.g., I-19), provide a marked selectivity over mTORC2 asmeasured by the impact of pAKT.

The location of this substitution is also critical to the observedselectivity. Introduction of larger substitutions at position 43 forexample does not lead to this unique selectivity profile claimed in thisapplication.

For the purpose of clarity, the structure of Rapamycin is reproducedbelow with the C-7 and C-43 positions noted.

In some embodiments, the present invention provides novel rapamycinanalogues that are potent mTORC1 inhibitors as measured by pS6K. UnlikeRapamycin and Everolimus, these compounds do not inhibit pAKT at longertime points (e.g., 24 hours and 48 hours). These compounds also showimproved solubility and improved pharmacokinetics comparing toRapamycin.

The activity of a compound utilized in this invention as an inhibitor ofmTORC1, may be assayed in vitro, in vivo or in a cell line. In vitroassays include assays that determine the inhibition of mTORC1. Detailedconditions for assaying a compound utilized in this invention as aninhibitor of mTORC1 are well known to one of ordinary skill in the art.Such methods are described in detail by Liu et al., Cancer Research,73(8): 2574-86 (2013) and Liu et al., J. Biological Chemistry 287(13):9742-52 (2012).

SUMMARY OF THE INVENTION

It has now been found that compounds of this invention, andpharmaceutically acceptable compositions thereof, are effective asinhibitors mTORC1 inhibitors. Such compounds have the general Formula I:

or a pharmaceutically acceptable salt thereof, wherein Ring A and R¹ areas defined and described herein.

Compounds of the present invention, and pharmaceutically acceptablecompositions thereof, are useful for treating a variety of diseases,disorders or conditions, associated with mTORC1. Such diseases,disorders, or conditions include those described herein.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows a comparison between two Western blots performed aftertreating PC3 cells with rapamycin or a compound of the present invention(I-29) for 24 and 48 hours. Staining indicates strong inhibition of themTORC1 pathway for both rapamycin and I-29 at both time points. Incontrast, the mTORC2 pathway, while inhibited by rapamycin at both 24and 48 hours, is not inhibited by I-29, as demonstrated by the lack ofAkt phosphorylation inhibition.

FIG. 2 shows a comparison among three Western blots performed aftertreating PC3 cells with rapamycin or a compound of the present inventionI-29 for 30, 15, or 5 minutes. Staining indicates a time dependentinhibition of the mTORC1 pathway for both rapamycin and I-29.

FIG. 3 shows a Western blot performed after treating PC3 cells withrapamycin, temsirolimus, everolimus, ridaforolimus, or a compound of thepresent invention (I-118) for 24 hrs. Staining indicates stronginhibition of the mTORC1 pathway for all compounds, and moderateconcentration dependent inhibition of mTORC1 by I-118. Significantly,rapamycin, temsirolimus, everolimus, and ridaforolimus show a dosedependent inhibition of the mTORC2 pathway, while I-118 does not inhibitthe mTORC2 pathway, as demonstrated by the lack of Akt phosphorylationinhibition.

FIG. 4 shows a Western blot performed after treating PC3 cells withrapamycin or compounds of the present invention (I-106, I-113, andI-118) for 24 hours. Staining indicates strong inhibition of the mTORC1pathway for each compound tested and no inhibition of 4E-BP1phosphorylation. Significantly, the compounds of the present inventiondo not inhibit mTORC2, as demonstrated by the lack of Aktphosphorylation inhibition.

FIG. 5 shows a Western blot performed after treating PC3 cells withrapamycin or compounds of the present invention (I-117, I-102, I-103,and I-39) for 24 hours. Staining indicates strong inhibition of themTORC1 pathway for each compound tested and no inhibition of 4E-BP1phosphorylation. Significantly, the compounds of the present inventiondo not inhibit mTORC2, as demonstrated by the lack of Aktphosphorylation inhibition.

FIG. 6 shows a Western blot performed after treating PC3 cells withrapamycin or compounds of the present invention (I-117, I-99, I-100, andI-101) for 24 hours. Staining indicates strong inhibition of the mTORC1pathway for rapamycin I-117, I-100, and I-101, and an appreciableconcentration dependent inhibition of the mTORC1 pathway for I-99.Significantly, the compounds of the present invention do not inhibitmTORC2, as demonstrated by the lack of Akt phosphorylation inhibition.

FIG. 7 shows a Western blot performed after treating PC3 cells withrapamycin or a compound of the present invention (I-117) for 24.Staining indicates strong inhibition of the mTOCR1 pathway by bothcompounds tested. Significantly, the compound of the present inventiondoes not inhibit mTORC2, as demonstrated by the lack of Aktphosphorylation inhibition.

FIG. 8 shows a Western blot performed after treating PC3 cells withrapamycin or compounds of the present invention (I-39, I-101, and I-99)for 24 hours. Staining indicates strong inhibition of the mTORC1 pathwayfor rapamycin and I-39, and an appreciable concentration dependentinhibition of the mTORC1 pathway for I-101 and I-99. Significantly, thecompounds of the present invention do not inhibit mTORC2, asdemonstrated by the lack of Akt phosphorylation inhibition.

FIG. 9 shows a Western blot performed after treating PC3 cells withrapamycin or compounds of the present invention (I-98 and I-97) for 24hours. Staining indicates strong inhibition of the mTORC1 pathway forrapamycin and I-98, and a moderate concentration dependent inhibition ofthe mTORC1 pathway for I-101 and I-99. Significantly, the compounds ofthe present invention do not inhibit mTORC2, as demonstrated by the lackof Akt phosphorylation inhibition.

FIG. 10 shows a Western blot performed after treating PC3 cells withrapamycin or compounds of the present invention (I-96 and I-100) for 24hours. Staining indicates strong inhibition of the mTORC1 pathway forall compounds tested. Significantly, the compounds of the presentinvention do not inhibit mTORC2, as demonstrated by the lack of Aktphosphorylation inhibition.

FIG. 11 shows a Western blot performed after treating PC3 cells withrapamycin, everolimus, or a compound of the present invention (I-7) for90 minutes. Staining indicates strong inhibition of the mTORC1 pathwayfor rapamycin and everolimus, and a moderate concentration dependentinhibition of the mTORC1 pathway for I-7. Significantly, the compound ofthe present invention does not inhibit mTORC2, as demonstrated by thelack of Akt phosphorylation inhibition.

FIG. 12 shows a Western blot performed after treating PC3 cells withrapamycin, everolimus, or a compound of the present invention (I-7) for24 hours. Staining indicates strong inhibition of the mTORC1 pathway forrapamycin and everolimus, and a moderate concentration dependentinhibition of the mTORC1 pathway for I-7. Significantly, the compound ofthe present invention does not inhibit mTORC2, as demonstrated by thelack of Akt phosphorylation inhibition.

FIG. 13 shows two Western blots performed after treating Jurkat cellswith everolimus or compounds of the present invention (I-29 and I-117)for 24 hrs. Staining indicates strong inhibition of the mTORC1 pathwayfor all compounds tested. Significantly, the compounds of the presentinvention do not inhibit mTORC2, as demonstrated by the lack of Aktphosphorylation inhibition.

FIG. 14 shows two Western blots performed after treating tuberoussclerosis (TSC2) negative (TSC−/−) MEF cells with everolimus orcompounds of the present invention (I-29, I-7, and I-117) for 90minutes. Staining indicates strong inhibition of the mTORC1 pathway foreverolimus, I-29, and I-117, and appreciable concentration dependentinhibition of the mTORC1 pathway for I-7. Significantly, the compoundsof the present invention do not inhibit mTORC2, as demonstrated by thelack of Akt phosphorylation inhibition.

FIG. 15 shows two Western blots performed after treating tuberoussclerosis 2 (TSC2) positive (TSC+/+) MEF cells with everolimus orcompounds of the present invention (I-29, I-7, and I-117) for 90minutes. Staining indicates strong inhibition of the mTORC1 pathway foreverolimus and I-29, moderate concentration dependent inhibition of themTORC1 pathway for I-117, and appreciable concentration dependentinhibition of the mTORC1 pathway for I-7. Significantly, the compoundsof the present invention do not inhibit mTORC2, as demonstrated by thelack of Akt phosphorylation inhibition.

FIG. 16 shows two Western blots performed after treating TSC−/−MEF cellswith everolimus or compounds of the present invention (I-29, I-7, andI-117) for 24 hours. Staining indicates strong inhibition of the mTORC1pathway for everolimus, I-29, and I-117, and an appreciableconcentration dependent inhibition of the mTORC1 pathway for I-7.Significantly, the compounds of the present invention do not inhibitmTORC2, as demonstrated by the lack of Akt phosphorylation inhibition.

FIG. 17 shows two Western blots performed after treating TSC+/+MEF cellswith everolimus or compounds of the present invention (I-29 and I-7) for24 hours. Staining indicates strong inhibition of the mTORC1 pathway foreverolimus, a moderate concentration dependent inhibition of the mTORC1pathway for I-29, and a modest concentration dependent inhibition of themTORC1 pathway for I-7. Significantly, the compounds of the presentinvention do not inhibit mTORC2, as demonstrated by the lack of Aktphosphorylation inhibition.

FIG. 18 shows a Western blot performed after treating TSC−/−MEF cellswith everolimus, rapamycin, or compounds of the present invention (I-2and I-92) for 24 hours. Staining indicates strong inhibition of themTORC1 pathway for everolimus, rapamycin, and I-2, and a moderateconcentration dependent inhibition of the mTORC1 pathway for I-92.

FIG. 19 shows a Western blot performed after treating TSC+/+MEF cellswith everolimus, rapamycin, or compounds of the present invention (I-2and I-92) for 24 hours. Staining indicates strong inhibition of themTORC1 pathway for everolimus, rapamycin, and moderate concentrationdependent inhibition of the mTORC1 pathway for I-2 and I-92.Significantly, the compounds of the present invention do not inhibitmTORC2, as demonstrated by the lack of Akt phosphorylation inhibition.

FIG. 20 shows a Western blot performed after treating TSC+/+MEF cellswith everolimus, or compounds of the present invention (I-20, I-29 andI-36) for 24 hours. Staining indicates strong inhibition of the mTORC1pathway for everolimus, and moderate concentration dependent inhibitionof the mTORC1 pathway for I-20, I-29, and I-36. Significantly, thecompounds of the present invention do not inhibit mTORC2, asdemonstrated by the lack of Akt phosphorylation inhibition.

FIG. 21 shows a Western blot performed after treating TSC+/+MEF cellswith everolimus, or compounds of the present invention (I-35, I-7 andI-26) for 24 hours. Staining indicates strong inhibition of the mTORC1pathway for everolimus, and modest concentration dependent inhibition ofthe mTORC1 pathway for I-35, I-7, and I-26. Significantly, the compoundsof the present invention do not inhibit mTORC2, as demonstrated by thelack of Akt phosphorylation inhibition.

FIG. 22 shows a Western blot performed after treating TSC+/+MEF cellswith everolimus, or compounds of the present invention (I-9, I-97 andI-98) for 24 hours. Staining indicates strong inhibition of the mTORC1pathway for everolimus, modest concentration dependent inhibition of themTORC1 pathway for I-9, I-97, and I-98. Significantly, the compounds ofthe present invention do not inhibit mTORC2, as demonstrated by the lackof Akt phosphorylation inhibition.

FIG. 23 shows a Western blot performed after treating TSC+/+MEF cellswith everolimus, or a compounds of the present invention (I-91) for 90minutes. Staining indicates strong inhibition of the mTORC1 pathway forrapamycin and I-91. Interestingly, I-91 exhibits some inhibition ofmTORC2, as demonstrated by Akt phosphorylation inhibition.

FIG. 24 shows a Western blot performed after treating TSC+/+MEF cellswith everolimus, or a compounds of the present invention (I-91) for 24hours. Staining indicates strong inhibition of the mTORC1 pathway forrapamycin and I-91. Interestingly, I-91 exhibits some inhibition ofmTORC2, as demonstrated by Akt phosphorylation inhibition.

FIG. 25 shows a Western blot performed after treating TSC+/+MEF cellswith everolimus, or a compounds of the present invention (I-105) for 90minutes. Staining indicates strong inhibition of the mTORC1 pathway forrapamycin and I-105. Significantly, the compound of the presentinvention does not inhibit mTORC2, as demonstrated by the lack of Aktphosphorylation inhibition.

FIG. 26 shows a Western blot performed after treating TSC+/+MEF cellswith everolimus, or a compounds of the present invention (I-105) for 24hours. Staining indicates strong inhibition of the mTORC1 pathway forrapamycin and I-105. Significantly, the compound of the presentinvention does not inhibit mTORC2, as demonstrated by the lack of Aktphosphorylation inhibition.

FIG. 27 shows two Western blots performed after treating PC3 cells witheverolimus or compounds of the present invention (I-2 and I-92) for 24hours. Staining indicates strong inhibition of the mTORC1 pathway foreach compound tested and no inhibition of 4E-BP1 phosphorylation.Significantly, the compounds of the present invention do not inhibitmTORC2, as demonstrated by the lack of Akt phosphorylation inhibition.

FIG. 28 shows a Western blot performed after treating TSC+/+MEF cellswith everolimus or a compound of the present invention (I-105) for 24hours. Staining indicates strong inhibition of the mTORC1 pathway foreach compound tested and no inhibition of 4E-BP1 phosphorylation.Significantly, the compounds of the present invention do not inhibitmTORC2, as demonstrated by the lack of Akt phosphorylation inhibition.

FIG. 29 shows a Western blot performed after treating TSC+/+MEF cellswith everolimus or compounds of the present invention (I-90 and I-110)for 24 hours. Staining indicates strong inhibition of the mTORC1 pathwayfor everolimus and I-90, and moderate concentration dependent inhibitionof the mTORC1 pathway for I-110. Significantly, the compounds of thepresent invention do not inhibit mTORC2, as demonstrated by the lack ofAkt phosphorylation inhibition.

FIG. 30 shows a Western blot performed after treating TSC+/+MEF cellswith everolimus or a compound of the present invention (I-115) for 24hours. Staining indicates strong inhibition of the mTORC1 pathway foreverolimus and moderate concentration dependent inhibition of the mTORC1pathway for I-115. Significantly, the compound of the present inventiondoes not inhibit mTORC2, as demonstrated by the lack of Aktphosphorylation inhibition.

FIG. 31 shows a Western blot performed after treating wild-type MEFcells with everolimus or compounds of the present invention (I-105,I-117, I-29, and I-90) for 24 hours. Staining indicates stronginhibition of the mTORC1 pathway for everolimus, and moderate inhibitionof the mTORC1 pathway for I-105, I-117, I-29, and I-90. Significantly,the compounds of the present invention do not inhibit mTORC2, asdemonstrated by the lack of Akt phosphorylation inhibition.

FIG. 32 shows a Western blot performed after treating wild-type MEFcells with everolimus or compounds of the present invention (I-105,I-117, I-29, and I-90) for 24 hours. Staining indicates stronginhibition of the mTORC1 pathway for each compound tested.Significantly, the compounds of the present invention do not inhibitmTORC2, as demonstrated by the lack of Akt phosphorylation inhibition.

FIG. 33 shows a Western blot performed after treating wild-type MEFcells with everolimus or compounds of the present invention (I-85 andI-83) for 90 minutes. Staining indicates strong inhibition of the mTORC1pathway for each compound tested. Significantly, the compounds of thepresent invention do not inhibit mTORC2, as demonstrated by the lack ofAkt phosphorylation inhibition.

FIG. 34 shows a Western blot performed after treating wild-type MEFcells with everolimus or compounds of the present invention (I-85 andI-83) for 24 hours. Staining indicates strong inhibition of the mTORC1pathway for each compound tested. Significantly, the compounds of thepresent invention do not inhibit mTORC2, as demonstrated by the lack ofAkt phosphorylation inhibition.

FIG. 35 shows a Western blot performed after treating PC3 cells witheverolimus or compounds of the present invention (I-85 and I-83) for 24hours. Staining indicates strong inhibition of the mTORC1 pathway foreach compound tested.

FIG. 36 shows a Western blot performed after treating PC3 cells andwild-type MEF cells with everolimus or a compound of the presentinvention (I-115) for 24 hours. Staining indicates strong inhibition ofthe mTORC1 pathway for each compound tested in the PC3 cells, stronginhibition of the mTORC1 pathway for everolimus in WT MEF cells, andmoderate concentration dependent inhibition of the mTORC1 pathway forI-115 in WT MEF cells. Significantly, the compound of the presentinvention does not inhibit mTORC2, as demonstrated by the lack of Aktphosphorylation inhibition.

FIG. 37 shows a Western blot performed after treating PC3 cells witheverolimus, a compound of the present invention (I-117), short PEGs,everolimus in combination with a short PEG, or I-117 in combination witha short PEG, for 24 hours. Staining indicates strong inhibition of themTORC1 pathway for everolimus and I-117, alone and in combination with ashort PEG. The short PEGs did not alone show inhibition of mTORC1 ormTORC2. Significantly, the compound of the present invention does notinhibit mTORC2, either alone or in combination with a short PEG, asdemonstrated by the lack of Akt phosphorylation inhibition.

FIG. 38 shows a Western blot performed after treating PC3 cells witheverolimus or compounds of the present invention (I-71, I-73 and I-75)for 24 hours. Staining indicates strong inhibition of the mTORC1 pathwayfor all compounds tested. Significantly, the compounds of the presentinvention do not inhibit mTORC2, as demonstrated by the lack of Aktphosphorylation inhibition.

FIG. 39 shows a Western blot performed after treating PC3 cells witheverolimus or compounds of the present invention (I-85 and I-83) for 24hours. Staining indicates strong inhibition of the mTORC1 pathway forall compounds tested. Significantly, the compounds of the presentinvention do not inhibit mTORC2, as demonstrated by the lack of Aktphosphorylation inhibition.

FIG. 40 shows a Western blot performed after treating PC3 cells witheverolimus or a compounds of the present invention (I-65) for 24 hours.Staining indicates strong inhibition of the mTORC1 pathway for bothcompounds tested. Significantly, the compound of the present inventiondoes not inhibit mTORC2, as demonstrated by the lack of Aktphosphorylation inhibition.

FIG. 41 shows a Western blot performed after treating PC3 cells witheverolimus or compounds of the present invention (I-5, I-106 and I-102)for 24 hours. Staining indicates strong inhibition of the mTORC1 pathwayfor all compounds tested. Significantly, the compounds of the presentinvention do not inhibit mTORC2, as demonstrated by the lack of Aktphosphorylation inhibition.

FIG. 42 shows a Western blot performed after treating PC3 cells witheverolimus or compounds of the present invention (I-75, I-71 and I-62)for 24 hours. Staining indicates strong inhibition of the mTORC1 pathwayfor all compounds tested. Significantly, the compounds of the presentinvention do not inhibit mTORC2, as demonstrated by the lack of Aktphosphorylation inhibition.

FIG. 43 shows the time course for a glucose tolerance and insulinsensitivity test in lean C57Bl/6 mice.

FIG. 44 shows the results of an intraperitoneal glucose tolerance testin lean C57Bl/6 mice during chronic treatment with rapamycin, I-29 orvehicle. ***P<0.001; **** P<0.0001; one-ANOVA and all bars indicate meanand SD.

FIG. 45 shows the area under the curve (AUC) for glucose clearance inlean C57Bl/6 mice during chronic treatment with rapamycin, I-29 orvehicle. ***P<0.001; T-test and all bars indicate mean and SD.

FIG. 46 shows Sirius red staining of kidney tissue from an AKI/CKD mousemodel.

FIG. 47 shows the percent area of kidney tissue fibrosis in an AKI/CKDmouse model following treatment with everolimus, I-29, I-117, orvehicle. *P=0.02 compared with vehicle, t-test.

FIG. 48 shows the collagen I mRNA expression in an AKI/CKD mouse modelfollowing treatment with vehicle or I-29. **P<0.01, ***P<0.001 vs. sham;††P<0.01 vs. vehicle.

FIG. 49 shows the collagen III mRNA expression in an AKI/CKD mouse modelfollowing treatment with vehicle or I-29. **P<0.01 vs. sham; †P<0.05 vs.vehicle.

FIG. 50 shows the fibronectin mRNA expression in an AKI/CKD mouse modelfollowing treatment with vehicle or I-29. **P<0.01 vs. sham; †P<0.05 vs.vehicle.

FIG. 51 shows the area of kidney tissue infiltrated by macrophages in anAKI/CKD mouse model following treatment with vehicle or I-29. *P<0.05,***P<0.001 vs. sham; ††P<0.05 vs. vehicle.

FIG. 52 shows the percent inhibition of IFN-γ production in anallogeneic MLR assay following treatment with rapamycin, everolimus,I-29, or I-117.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS 1. General Description ofCertain Embodiments of the Invention

In certain embodiments, the present invention provides a compound ofFormula I:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   Ring A is a monovalent derivative of rapamycin or an analog        (i.e., a rapalog) thereof,        -   wherein R¹ is attached thereto at the C-7 hydroxyl position            of the rapamycin, or analog thereof;    -   R¹ is an optionally substituted straight or branched saturated        or unsaturated monovalent C₃₋₃₀ hydrocarbon chain wherein one or        more methylene units of R¹ are optionally and independently        replaced by —N(R)—, —N(R)C(O)—, —C(O)N(R)—, —N(R)S(O)₂—,        —S(O)₂N(R)—, —O—, —C(O)—, —OC(O)—, —C(O)O—, —S—, —S(O)—,        —S(O)₂—, or —P(O)(R)₂, or a 6-18 membered saturated or partially        unsaturated heterocyclic ring having I-6 heteroatoms        independently selected from nitrogen, oxygen, or sulfur; or        -   R¹ is selected from Formula P-0:

-   -   -   wherein:            -   indicates the attachment point to Ring A;            -   each Z is independently —O—, —S—, —NR—, or —SO₂—;            -   n is from about 2 to about 300; and

    -   each R is independently hydrogen or an optionally substituted        C₁₋₆ aliphatic group.

In some embodiments, the present invention provides a compound ofFormula I other than those selected from:

2. Compounds and Definitions

Compounds of the present invention include those described generallyherein, and are further illustrated by the classes, subclasses, andspecies disclosed herein. As used herein, the following definitionsshall apply unless otherwise indicated. For purposes of this invention,the chemical elements are identified in accordance with the PeriodicTable of the Elements, CAS version, Handbook of Chemistry and Physics,75^(th) Ed. Additionally, general principles of organic chemistry aredescribed in “Organic Chemistry”, Thomas Sorrell, University ScienceBooks, Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5^(th)Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001,the entire contents of which are hereby incorporated by reference.

The term “aliphatic” or “aliphatic group”, as used herein, means astraight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbonor bicyclic hydrocarbon that is completely saturated or that containsone or more units of unsaturation, but which is not aromatic (alsoreferred to herein as “carbocycle,” “cycloaliphatic” or “cycloalkyl”),that has a single point of attachment to the rest of the molecule.Unless otherwise specified, aliphatic groups contain 1-6 aliphaticcarbon atoms. In some embodiments, aliphatic groups contain 1-5aliphatic carbon atoms. In other embodiments, aliphatic groups contain1-4 aliphatic carbon atoms. In still other embodiments, aliphatic groupscontain 1-3 aliphatic carbon atoms, and in yet other embodiments,aliphatic groups contain 1-2 aliphatic carbon atoms. In someembodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refersto a monocyclic C₃-C₆ hydrocarbon that is completely saturated or thatcontains one or more units of unsaturation, but which is not aromatic,that has a single point of attachment to the rest of the molecule.Suitable aliphatic groups include, but are not limited to, linear orbranched, substituted or unsubstituted alkyl, alkenyl, alkynyl groupsand hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or(cycloalkyl)alkenyl.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon (including, any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen or; a substitutable nitrogen of a heterocyclic ring, forexample N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR⁺ (as in N-substituted pyrrolidinyl)).

The term “unsaturated,” as used herein, means that a moiety has one ormore units of unsaturation.

As used herein, the term “bivalent C₁₋₈ (or C₁₋₆) saturated orunsaturated, straight or branched, hydrocarbon chain”, refers tobivalent alkylene, alkenylene, and alkynylene chains that are straightor branched as defined herein.

The term “alkylene” refers to a bivalent alkyl group. An “alkylenechain” is a polymethylene group, i.e., —(CH₂)_(n)—, wherein n is apositive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylenegroup in which one or more methylene hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

The term “alkenylene” refers to a bivalent alkenyl group. A substitutedalkenylene chain is a polymethylene group containing at least one doublebond in which one or more hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

The term “halogen” means F, Cl, Br, or I.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic orbicyclic ring systems having a total of five to fourteen ring members,wherein at least one ring in the system is aromatic and wherein eachring in the system contains 3 to 7 ring members. The term “aryl” may beused interchangeably with the term “aryl ring.” In certain embodimentsof the present invention, “aryl” refers to an aromatic ring system whichincludes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl andthe like, which may bear one or more substituents. Also included withinthe scope of the term “aryl,” as it is used herein, is a group in whichan aromatic ring is fused to one or more non-aromatic rings, such asindanyl, phthalimidyl, naphthimidyl, phenanthridinyl, ortetrahydronaphthyl, and the like.

The terms “heteroaryl” and “heteroar-,” used alone or as part of alarger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,” refer togroups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms;having 6, 10, or 14 π electrons shared in a cyclic array; and having, inaddition to carbon atoms, from one to five heteroatoms. The term“heteroatom” refers to nitrogen, oxygen, or sulfur, and includes anyoxidized form of nitrogen or sulfur, and any quaternized form of a basicnitrogen. Heteroaryl groups include, without limitation, thienyl,furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, and pteridinyl. The terms “heteroaryl” and“heteroar-”, as used herein, also include groups in which aheteroaromatic ring is fused to one or more aryl, cycloaliphatic, orheterocyclyl rings, where the radical or point of attachment is on theheteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl,benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl,benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Aheteroaryl group may be mono- or bicyclic. The term “heteroaryl” may beused interchangeably with the terms “heteroaryl ring,” “heteroarylgroup,” or “heteroaromatic,” any of which terms include rings that areoptionally substituted. The term “heteroaralkyl” refers to an alkylgroup substituted by a heteroaryl, wherein the alkyl and heteroarylportions independently are optionally substituted.

As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclicradical,” and “heterocyclic ring” are used interchangeably and refer toa stable 5- to 7-membered monocyclic or 7-10-membered bicyclicheterocyclic moiety that is either saturated or partially unsaturated,and having, in addition to carbon atoms, one or more, preferably one tofour, heteroatoms, as defined above. When used in reference to a ringatom of a heterocycle, the term “nitrogen” includes a substitutednitrogen. As an example, in a saturated or partially unsaturated ringhaving 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, thenitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as inpyrrolidinyl), or ⁺NR (as in N-substituted pyrrolidinyl).

A heterocyclic ring can be attached to its pendant group at anyheteroatom or carbon atom that results in a stable structure and any ofthe ring atoms can be optionally substituted. Examples of such saturatedor partially unsaturated heterocyclic radicals include, withoutlimitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl,piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl,diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. Theterms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclicgroup,” “heterocyclic moiety,” and “heterocyclic radical,” are usedinterchangeably herein, and also include groups in which a heterocyclylring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings,such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, ortetrahydroquinolinyl. A heterocyclyl group may be mono- or bicyclic. Theterm “heterocyclylalkyl” refers to an alkyl group substituted by aheterocyclyl, wherein the alkyl and heterocyclyl portions independentlyare optionally substituted.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond. The term “partiallyunsaturated” is intended to encompass rings having multiple sites ofunsaturation, but is not intended to include aryl or heteroarylmoieties, as herein defined.

As described herein, compounds of the invention may contain “optionallysubstituted” moieties. In general, the term “substituted,” whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group may have a suitable substituent at each substitutable position ofthe group, and when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds. The term “stable,” as used herein, refers tocompounds that are not substantially altered when subjected toconditions to allow for their production, detection, and, in certainembodiments, their recovery, purification, and use for one or more ofthe purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(∘); —(CH₂)₀₋₄OR^(∘); —O(CH₂)₀₋₄R^(∘), —O—(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄CH(OR^(∘))₂; —(CH₂)₀₋₄SR^(∘); —(CH₂)₀₋₄Ph, which may besubstituted with R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(∘); —CH═CHPh, which may be substituted with R^(∘);—(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may be substituted with R^(∘); —NO₂;—CN; —N₃; —(CH₂)₀₋₄N(R^(∘))₂; —(CH₂)₀₋₄N(R^(∘))C(O)R^(∘);—N(R^(∘))C(S)R^(∘); —(CH₂)₀₋₄N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))C(S)NR^(∘)₂; —(CH₂)₀₋₄N(R^(∘))C(O)OR^(∘); —N(R^(∘))N(R^(∘))C(O)R^(∘);—N(R^(∘))N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))N(R^(∘))C(O)OR^(∘);—(CH₂)₀₋₄C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄C(O)SR^(∘); —(CH₂)₀₋₄C(O)OSiR^(∘) ₃; —(CH₂)₀₋₄OC(O)R^(∘);—OC(O)(CH₂)₀₋₄SR—, SC(S)SR^(∘); —(CH₂)₀₋₄SC(O)R^(∘); —(CH₂)₀₋₄C(O)NR^(∘)₂; —C(S)NR^(∘) ₂; —C(S)SR^(∘); —SC(S)SR^(∘), —(CH₂)₀₋₄OC(O)NR^(∘) ₂;—C(O)N(OR^(∘))R^(∘); —C(O)C(O)R^(∘); —C(O)CH₂C(O)R^(∘);—C(NOR^(∘))R^(∘); —(CH₂)₀₋₄SSR^(∘); —(CH₂)₀₋₄S(O)₂R^(∘);—(CH₂)₀₋₄S(O)₂OR^(∘); —(CH₂)₀₋₄OS(O)₂R^(∘); —S(O)₂NR^(∘) ₂;—(CH₂)₀₋₄S(O)R^(∘); —N(R^(∘))S(O)₂NR^(∘) ₂; —N(R^(∘))S(O)₂R^(∘);—N(OR^(∘))R^(∘); —C(NH)NR^(∘) ₂; —P(O)₂R^(∘); —P(O)R^(∘) ₂; —OP(O)R^(∘)₂; —OP(O)(OR^(∘))₂; SiR^(∘) ₃; —(C₁₋₄ straight or branchedalkylene)O—N(R^(∘))₂; or —(C₁₋₄ straight or branchedalkylene)C(O)O—N(R^(∘))₂, wherein each R^(∘) may be substituted asdefined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, —CH₂-(5-6 membered heteroaryl ring), or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(∘), taken together with their intervening atom(s), form a3-12-membered saturated, partially unsaturated, or aryl mono- orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(∘) (or the ring formed by takingtwo independent occurrences of R^(∘) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(●), -(haloR^(●)),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(●), —(CH₂)₀₋₂CH(OR^(●))₂; —O(haloR^(●)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(●), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(●),—(CH₂)₀₋₂SR^(●), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(●),—(CH₂)₀₋₂NR^(●) ₂, —NO₂, —SiR^(●) ₃, —OSiR^(●) ₃, —C(O)SR^(●), —(C₁₋₄straight or branched alkylene)C(O)OR^(●), or —SSR^(●) wherein each R^(●)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(∘) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(●) include halogen,—R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN, —C(O)OH,—C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein each R^(●) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein eachR^(†) is independently hydrogen, C₁₋₆ aliphatic which may be substitutedas defined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN,—C(O)OH, —C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein eachR^(●) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge etal., describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference. Pharmaceutically acceptable salts of the compounds of thisinvention include those derived from suitable inorganic and organicacids and bases. Examples of pharmaceutically acceptable, nontoxic acidaddition salts are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid and perchloric acid or with organic acids such as acetic acid,oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid ormalonic acid or by using other methods used in the art such as ionexchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate,propionate, stearate, succinate, sulfate, tartrate, thiocyanate,p-toluenesulfonate, undecanoate, valerate salts, and the like.

Salts derived from appropriate bases include alkali metal, alkalineearth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. Representative alkali oralkaline earth metal salts include sodium, lithium, potassium, calcium,magnesium, and the like. Further pharmaceutically acceptable saltsinclude, when appropriate, nontoxic ammonium, quaternary ammonium, andamine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and arylsulfonate.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, Z and E double bond isomers,and Z and E conformational isomers. Therefore, single stereochemicalisomers as well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures including the replacement of hydrogen by deuterium ortritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention. Such compounds are useful, forexample, as analytical tools, as probes in biological assays, or astherapeutic agents in accordance with the present invention.

The terms “measurable affinity” and “measurably inhibit,” as usedherein, means a measurable change in mTORC1 activity between a samplecomprising a compound of the present invention, or composition thereof,and mTORC1, and an equivalent sample comprising mTORC1 in the absence ofsaid compound, or composition thereof.

3. Description of Exemplary Embodiments

As described above, in certain embodiments, the present inventionprovides a compound of Formula I:

-   or a pharmaceutically acceptable salt thereof, wherein:    -   Ring A is a monovalent derivative of rapamycin or an analog        (i.e., a rapalog) thereof,        -   wherein R¹ is attached thereto at the C-7 hydroxyl position            of the rapamycin, or analog thereof; and    -   R¹ is an optionally substituted straight or branched saturated        or unsaturated monovalent C₃₋₃₀ hydrocarbon chain wherein one or        more methylene units of R¹ are optionally and independently        replaced by —N(R)—, —N(R)C(O)—, —C(O)N(R)—, —N(R)S(O)₂—,        —S(O)₂N(R)—, —O—, —C(O)—, —OC(O)—, —C(O)O—, —S—, —S(O)—,        —S(O)₂—, or —P(O)(R)₂; or a 6-18 membered saturated or partially        unsaturated heterocyclic ring having I-6 heteroatoms        independently selected from nitrogen, oxygen, or sulfur; or        -   R¹ is selected from Formula P-0:

-   -   -   wherein:            -   indicates the attachment point to Ring A;            -   each Z is independently —O—, —S—, —NR—, or —SO₂—;            -   n is from about 2 to about 300; and

    -   each R is independently hydrogen or an optionally substituted        C₁₋₆ aliphatic group.

It will be appreciated that the term “rapamycin”, and structuresthereof, recited throughout the specification is intended to encompassrapamycin and analogs thereof.

For the purpose of clarity, provided Formula II is reproduced below withthe R¹ moiety depicted attached at the C-7 hydroxyl position.Accordingly, the present invention provides a compound of Formula II:

-   or a pharmaceutically acceptable salt thereof, wherein:    -   R¹ is an optionally substituted straight or branched saturated        or unsaturated monovalent C₃₋₃₀ hydrocarbon chain wherein one or        more methylene units of R¹ are optionally and independently        replaced by —N(R)—, —N(R)C(O)—, —C(O)N(R)—, —N(R)S(O)₂—,        —S(O)₂N(R)—, —O—, —C(O)—, —OC(O)—, —C(O)O—, —S—, —S(O)—,        —S(O)₂—, or —P(O)(R)₂; or a 6-18 membered saturated or partially        unsaturated heterocyclic ring having I-6 heteroatoms        independently selected from nitrogen, oxygen, or sulfur; or        -   R¹ is selected from Formula P-0:

-   -   -   wherein:            -   indicates the attachment point to the C-7 hydroxyl                position;            -   each Z is independently —O—, —S—, —NR—, or —SO₂—;            -   n is from about 2 to about 300; and

    -   each R is independently hydrogen or an optionally substituted        C₁₋₆ aliphatic group.

In some embodiments, the present invention provides a compound ofFormula II-a or II-b:

-   or a pharmaceutically acceptable salt thereof, wherein each of R¹ is    as defined above and described herein in classes and subclasses.

It will be appreciated that the term “rapamycin”, and structure thereof,recited throughout the specification is intended to encompass rapamycinand analogs thereof. Accordingly, in certain embodiments Ring A isRapamycin. In some embodiments, Ring A is Everolimus. In someEmbodiments, Ring A is Temsirolimus. In some embodiments, Ring A isRidaforolimus. In some embodiments, Ring A is Umirolimus.

The above recited analogs of rapamycin (i.e., rapalogs) are forexemplification and not intended to limit the current invention.

As defined above, R¹ is an optionally substituted straight or branchedsaturated or unsaturated monovalent C₃₋₃₀ hydrocarbon chain wherein oneor more methylene units of R¹ are optionally and independently replacedby —N(R)—, —N(R)C(O)—, —C(O)N(R)—, —N(R)S(O)₂—, —S(O)₂N(R)—, —O—,—C(O)—, —OC(O)—, —C(O)O—, —S—, —S(O)—, or —S(O)₂—, or a 6-18 saturatedor partially unsaturated heterocyclic ring having 1-6 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, R¹ is an optionally substituted straight orbranched saturated or unsaturated monovalent C₃₋₃₀ hydrocarbon chainwherein one or more methylene units of R¹ are optionally andindependently replaced by —N(R)—, —N(R)C(O)—, —C(O)N(R)—, —N(R)S(O)₂—,—S(O)₂N(R)—, —O—, —C(O)—, —OC(O)—, —C(O)O—, —S—, —S(O)—, —S(O)₂—, or—P(O)(R)₂. In some embodiments, R¹ is an optionally substituted branchedsaturated monovalent hydrocarbon chain wherein one or more methyleneunits of R¹ are optionally and independently replaced by —N(R)—,—N(R)C(O)—, —C(O)N(R)—, —N(R)S(O)₂—, —S(O)₂N(R)—, —O—, —C(O)—, —OC(O)—,—C(O)O—, —S—, —S(O)—, —S(O)₂—, or —P(O)(R)₂. In some embodiments, R¹ isan optionally substituted straight unsaturated monovalent hydrocarbonchain wherein one or more methylene units of R¹ are optionally andindependently replaced by —N(R)—, —N(R)C(O)—, —C(O)N(R)—, —N(R)S(O)₂—,—S(O)₂N(R)—, —O—, —C(O)—, —OC(O)—, —C(O)O—S—, —S(O)—, —S(O)₂—, or—P(O)(R)₂. In some embodiments, R¹ is an optionally substituted branchedunsaturated monovalent hydrocarbon chain wherein one or more methyleneunits of R¹ are optionally and independently replaced by —N(R)—,—N(R)C(O)—, —C(O)N(R)—, —N(R)S(O)₂—, —S(O)₂N(R)—, —O—, —C(O)—, —OC(O)—,—C(O)O—, —S—, —S(O)—, —S(O)₂—, or —P(O)(R)₂. In some embodiments, R¹ isan optionally substituted straight saturated monovalent hydrocarbonchain wherein one or more methylene units of R¹ are optionally andindependently replaced by —O—.

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹

In some embodiments, R¹ is

In some embodiments, R¹ is

In certain embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In certain embodiments, R¹ is of Formula P-0:

-   wherein:    -   indicates the attachment point to Ring A;    -   each Z is independently —O—, —S—, —NR—, or —SO₂—;    -   n is from about 2 to about 300; and    -   each R is independently hydrogen, or an optionally substituted        C₁₋₆ aliphatic group.

In some embodiments, n is from about 2 to about 10; from about 10 toabout 20; from about 20 to about 30; from about 30 to about 40; fromabout 40 to about 50; from about 50 to about 60; from about 60 to about70; from about 70 to about 80; from about 80 to about 90; from about 90to about 100; from about 110 to about 120; from about 120 to about 130;from about 140 to about 150; from about 150 to about 160; from about 170to about 180; from about 180 to about 190; from about 190 to about 200;from 200 to about 210; from about 210 to about 220; from about 220 toabout 230; from about 230 to about 240; from about 240 to about 250;from about 250 to about 260; from about 260 to about 270; from about 270to about 280; from about 280 to about 290; or from about 290 to about300.

In some embodiments R¹ is

wherein n and R are as described herein.

In some embodiments, R¹ is

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In some embodiments, R¹ is

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In some embodiments, R¹ is

In some embodiments R¹ is

In certain embodiments, R¹ is a 6-18 membered saturated or partiallyunsaturated heterocyclic ring having 1-6 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is selected from those depicted in Table 1,below.

In certain embodiments, R is independently hydrogen or an optionallysubstituted group selected from C₁₋₆ aliphatic. In some embodiments, Ris hydrogen. In some embodiments, R is methyl. In some embodiments, R isethyl. In some embodiments, R is propyl. In some embodiments, R is

In some embodiments, R is selected from those depicted in Table 1,below.

In some embodiments, the present invention provides a compound ofFormula III:

-   or a pharmaceutically acceptable salt thereof, wherein:    -   R¹ is as described herein.

In some embodiments, the present invention provides a compound ofFormula III-a or III-b:

-   or a pharmaceutically acceptable salt thereof, wherein:    -   R¹ is as described herein.

In some embodiments, the present invention provides a compound ofFormula IV:

-   or a pharmaceutically acceptable salt thereof, wherein:    -   R¹ is as described herein.

In some embodiments, the present invention provides a compound ofFormula IV-a or IV-b:

-   or a pharmaceutically acceptable salt thereof, wherein:    -   R¹ is as described herein.

In some embodiments, the present invention provides a compound ofFormula V:

-   or a pharmaceutically acceptable salt thereof, wherein:    -   R¹ is as described herein.

In some embodiments, the present invention provides a compound ofFormula V-a or V-b:

-   or a pharmaceutically acceptable salt thereof, wherein:    -   R¹ is as described herein.

In some embodiments, the present invention provides a compound ofFormula VI:

-   or a pharmaceutically acceptable salt thereof, wherein:    -   R¹ is as described herein.

In some embodiments, the present invention provides a compound ofFormula VI-a or VI-b:

-   or a pharmaceutically acceptable salt thereof, wherein:    -   R¹ is as described herein.

In some embodiments, the present invention provides a compound ofFormula VII:

-   or a pharmaceutically acceptable salt thereof, wherein:    -   R¹ is as described herein.

In some embodiments, the present invention provides a compound ofFormula VII-a or VII-b:

-   or a pharmaceutically acceptable salt thereof, wherein:    -   R¹ is as described herein.

In some embodiments, the present invention provides a compound ofFormula VIII:

-   or a pharmaceutically acceptable salt thereof, wherein:    -   R¹ is as described herein.

In some embodiments, the present invention provides a compound ofFormula VIII-a or VIII-b:

-   or a pharmaceutically acceptable salt thereof, wherein:    -   R¹ is as described herein.

In some embodiments, the present invention provides a compound ofFormula IX-a, IX-b, IX-c, IX-d, IX-e, IX-f, or IX-g:

-   or a pharmaceutically acceptable salt thereof, wherein:    -   R¹ is as described herein.

In some embodiments, the present invention provides a compound ofFormula X-a, X-b, X-c, X-d, X-e, X-f, or X-g:

-   or a pharmaceutically acceptable salt thereof, wherein:    -   R¹ is as described herein.

In some embodiments, the present invention provides a compound ofFormula XI-a, XI-b, XI-c, XI-d, XI-e, XI-f, or XI-g:

-   or a pharmaceutically acceptable salt thereof, wherein:    -   P-0 is as described herein.

In some embodiments, the present invention provides a compound ofFormula XII:

-   or a pharmaceutically acceptable salt thereof, wherein:    -   R and n are as described herein.

In some embodiments, the present invention provides a compound ofFormulae XII-a or XII-b:

-   or a pharmaceutically acceptable salt thereof, wherein:    -   R and n are as described herein.

In some embodiments, the present invention provides a compound ofFormula XIII:

-   or a pharmaceutically acceptable salt thereof, wherein:    -   R and n are as described herein.

In some embodiments, the present invention provides a compound ofFormula XIII-a or XIII-b:

-   or a pharmaceutically acceptable salt thereof, wherein:    -   R and n are as described herein.

In some embodiments, the present invention provides a compound ofFormula XIV:

-   or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides a compound ofFormula XIV-a or XIV-b:

-   or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides a compound ofFormula XV:

-   or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides a compound ofFormula XV-a or XV-b:

-   -   or a pharmaceutically acceptable salt thereof.

Rapamycin is marketed under the brand name Rapamune® (generic name,sirolimus) and is well known for its antiproliferative andimmunosuppressive activity. Rapamycin is FDA approved for the preventionof transplant rejection and for coating stents to prevent restenosis.Aside from the documented benefits of rapamycin, it is well known thatrapamycin is associated with a number of serious side effects. Such sideeffects include diabetes-like symptoms of decreased glucose toleranceand lowering of insulin sensitivity. In addition, it has been reportedthat rapamycin activates the Akt signaling pathway (including activationof Akt and ERK) thereby increasing a patient's risk of cancer.

As used herein the phrase “rapamycin alone” is intended to compare acompound of the present invention with rapamycin, or an analog thereof,as alternatives.

In some embodiments, a provided compound of Formula I, II, III, IV, V,VI, VII, VIII, XII, XIII, XIV, or XV is more efficatious than rapamycinalone. In some embodiments, a provided compound of Formula II-a, III-a,IV-a, V-a, VI-a, VII-a, VIII-a, XII-a, XIII-a, XIV-a, or XV-a is moreefficatious than rapamycin alone. In some embodiments, a providedcompound of Formula II-b, III-b, IV-b, V-b, VI-b, VII-b, VIII-b, XII-b,XIII-b, XIV-b, or XV-b is more efficatious than rapamycin alone.

In some embodiments, a provided compound of Formula IX-a, IX-b, IX-c,IX-d, IX-e, IX-f, or IX-g is more efficatious than rapamycin alone.

In some embodiments, a provided compound of Formula X-a, X-b, X-c, X-d,X-e, X-f, or X-g is more efficatious than rapamycin alone.

In some embodiments, a provided compound of Formula XI-a, XI-b, XI-c,XI-d, XI-e, XI-f, or XI-g is more efficatious than rapamycin alone.

In some embodiments, a provided compound of Formula II-a or II-b is moreefficatious than rapamycin alone. In some embodiments, a providedcompound of Formula III-a or III-b is more efficatious than rapamycinalone. In some embodiments, a provided compound of Formula IV-a or IV-bis more efficatious than rapamycin alone. In some embodiments, aprovided compound of Formula V-a or V-b is more efficatious thanrapamycin alone. In some embodiments, a provided compound of FormulaVI-a or VI-b is more efficatious than rapamycin alone. In someembodiments, a provided compound of Formula VII-a or VII-b is moreefficatious than rapamycin alone. In some embodiments, a providedcompound of Formula VIII-a or VIII-b is more efficatious than rapamycinalone. In some embodiments, a provided compound of Formula XII-a orXII-b is more efficatious than rapamycin alone. In some embodiments, aprovided compound of Formula XIII-a or XIII-b is more efficatious thanrapamycin alone. In some embodiments, a provided compound of FormulaXIV-a or XIV-b is more efficatious than rapamycin alone. In someembodiments, a provided compound of Formula XV-a or XV-b is moreefficatious than rapamycin alone.

In some embodiments, a provided compound of Formula I, II, III, IV, V,VI, VII, VIII, XII, XIII, XIV, or XV when administered to a patient,results in fewer and/or lesser severity of side effects than whenrapamycin is administered.

In some embodiments, a provided compound of Formula IX-a, IX-b, IX-c,IX-d, IX-e, IX-f, or IX-g when administered to a patient, results infewer and/or lesser severity of side effects than when rapamycin isadministered.

In some embodiments, a provided compound of Formula X-a, X-b, X-c, X-d,X-e, X-f, or X-g when administered to a patient, results in fewer and/orlesser severity of side effects than when rapamycin is administered.

In some embodiments, a provided compound of Formula XI-a, XI-b, XI-c,XI-d, XI-e, XI-f, or XI-g when administered to a patient, results infewer and/or lesser severity of side effects than when rapamycin isadministered.

Exemplary compounds of the invention are set forth in Table 1, below.

TABLE 1 Exemplary Compounds

I-1

I-2

I-3

I-4

I-5

I-6

I-7

I-8

I-9

I-10

I-11

I-12

I-13

I-14

I-15

I-16

I-17

I-18

I-19

I-20

I-21

I-22

I-23

I-24

I-25

I-26

I-27

I-28

I-29

I-30

I-31

I-32

I-33

I-34

I-35

I-36

I-37

I-38

I-39

I-41

I-42

I-44

I-45

I-47

I-48

I-49

I-50

I-51

I-52

I-53

I-54

I-55

I-56

I-57

I-58

I-59

I-60

I-61

I-62

I-63

I-64

I-65

I-66

I-67

I-68

I-69

I-70

I-71

I-72

I-73

I-74

I-75

I-76

I-77

I-78

I-79

I-80

I-81

I-82

I-83

I-84

I-85

I-86

I-87

I-88

I-89

I-90

I-91

I-92

I-95

I-96

I-97

I-98

I-99

I-100

I-101

I-102

I-103

I-104

I-105

I-106

I-107

I-108

I-109

I-110

I-111

I-112

I-113

I-114

I-115

I-116

I-117

I-118

I-119

I-120

I-121

I-123

I-125

I-126

I-127

I-128

I-129

In some embodiments, the present invention provides a compound set forthin Table 1, above, or a pharmaceutically acceptable salt thereof. Itwill be appreciated that the present invention also provides a compoundset forth in Table 1, above, as a racemic mixture at the C7 position, ora pharmaceutically acceptable salt thereof. Further, it will beappreciated that compounds set forth in Table 1, above, as racemicmixtures at the C7 hydroxyl position may be separated into diastereomersby various methods, e.g., chiral chromatography.

4. Uses, Formulation and Administration

Pharmaceutically Acceptable Compositions

According to another embodiment, the invention provides a compositioncomprising a compound of this invention or a pharmaceutically acceptablederivative thereof and a pharmaceutically acceptable carrier, adjuvant,or vehicle. The amount of compound in compositions of this invention issuch that is effective to measurably inhibit mTORC1, in a biologicalsample or in a patient. In certain embodiments, the amount of compoundin compositions of this invention is such that is effective tomeasurably inhibit mTORC1, in a biological sample or in a patient. Incertain embodiments, a composition of this invention is formulated foradministration to a patient in need of such composition. In someembodiments, a composition of this invention is formulated for oraladministration to a patient.

The term “patient,” as used herein, means an animal, preferably amammal, and most preferably a human.

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle”refers to anon-toxic carrier, adjuvant, or vehicle that does not destroythe pharmacological activity of the compound with which it isformulated. Pharmaceutically acceptable carriers, adjuvants or vehiclesthat may be used in the compositions of this invention include, but arenot limited to, ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

Compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously. Sterile injectable forms of thecompositions of this invention may be aqueous or oleaginous suspension.These suspensions may be formulated according to techniques known in theart using suitable dispersing or wetting agents and suspending agents.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium.

For this purpose, any bland fixed oil may be employed includingsynthetic mono- or di-glycerides. Fatty acids, such as oleic acid andits glyceride derivatives are useful in the preparation of injectables,as are natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as carboxymethyl cellulose or similar dispersingagents that are commonly used in the formulation of pharmaceuticallyacceptable dosage forms including emulsions and suspensions. Othercommonly used surfactants, such as Tweens, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

Pharmaceutically acceptable compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers commonly used include lactose andcorn starch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. These can be prepared by mixing the agent with asuitable non-irritating excipient that is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

Pharmaceutically acceptable compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, provided pharmaceutically acceptablecompositions may be formulated in a suitable ointment containing theactive component suspended or dissolved in one or more carriers.Carriers for topical administration of compounds of this inventioninclude, but are not limited to, mineral oil, liquid petrolatum, whitepetrolatum, propylene glycol, polyoxyethylene, polyoxypropylenecompound, emulsifying wax and water. Alternatively, providedpharmaceutically acceptable compositions can be formulated in a suitablelotion or cream containing the active components suspended or dissolvedin one or more pharmaceutically acceptable carriers. Suitable carriersinclude, but are not limited to, mineral oil, sorbitan monostearate,polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol,benzyl alcohol and water.

For ophthalmic use, provided pharmaceutically acceptable compositionsmay be formulated as micronized suspensions in isotonic, pH adjustedsterile saline, or, preferably, as solutions in isotonic, pH adjustedsterile saline, either with or without a preservative such asbenzylalkonium chloride. Alternatively, for ophthalmic uses, thepharmaceutically acceptable compositions may be formulated in anointment such as petrolatum.

Pharmaceutically acceptable compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

Most preferably, pharmaceutically acceptable compositions of thisinvention are formulated for oral administration. Such formulations maybe administered with or without food. In some embodiments,pharmaceutically acceptable compositions of this invention areadministered without food. In other embodiments, pharmaceuticallyacceptable compositions of this invention are administered with food.

The amount of compounds of the present invention that may be combinedwith the carrier materials to produce a composition in a single dosageform will vary depending upon the host treated, the particular mode ofadministration. Preferably, provided compositions should be formulatedso that a dosage of between 0.01-100 mg/kg body weight/day of theinhibitor can be administered to a patient receiving these compositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a compound of the present invention in the composition willalso depend upon the particular compound in the composition.

Uses of Compounds and Pharmaceutically Acceptable Compositions

As used herein, the terms “treatment,” “treat,” and “treating” refer toreversing, alleviating, delaying the onset of, or inhibiting theprogress of a disease or disorder, or one or more symptoms thereof, asdescribed herein. In some embodiments, treatment may be administeredafter one or more symptoms have developed. In other embodiments,treatment may be administered in the absence of symptoms. For example,treatment may be administered to a susceptible individual prior to theonset of symptoms (e.g., in light of a history of symptoms and/or inlight of genetic or other susceptibility factors). Treatment may also becontinued after symptoms have resolved, for example to prevent or delaytheir recurrence.

Provided compounds are inhibitors of mTORC1 and are therefore useful fortreating one or more disorders associated with activity of mTORC1. Thus,in certain embodiments, the present invention provides a method fortreating an mTORC1-mediated disorder comprising the step ofadministering to a patient in need thereof a compound of the presentinvention, or pharmaceutically acceptable composition thereof.

As used herein, the terms “mTORC1-mediated” disorders, diseases, and/orconditions as used herein means any disease or other deleteriouscondition in which mTORC1, is known to play a role. Accordingly, anotherembodiment of the present invention relates to treating or lessening theseverity of one or more diseases in which mTORC1 is known to play arole. In certain embodiments, an mTORC1-mediated disorder, disease,and/or condition is selected from those described by Matt Kaeberlin,Scientifica, vol. 2013, Article ID 849186.

The methods described herein include methods for the treatment of cancerin a subject. As used in this context, to “treat” means to ameliorate orimprove at least one symptom or clinical parameter of the cancer. Forexample, a treatment can result in a reduction in tumor size or growthrate. A treatment need not cure the cancer or cause remission 100% ofthe time, in all subjects.

As used herein, the term “cancer” refers to cells having the capacityfor autonomous growth, i.e., an abnormal state or conditioncharacterized by rapidly proliferating cell growth. The term is meant toinclude all types of cancerous growths or oncogenic processes,metastatic tissues or malignantly transformed cells, tissues, or organs,irrespective of histopathologic type or stage of invasiveness. The term“tumor” as used herein refers to cancerous cells, e.g., a mass of cancercells.

Cancers that can be treated or diagnoses using the methods describedherein include malignancies of the various organ systems, such asaffecting lung, breast, thyroid, lymphoid, gastrointestinal, andgenito-urinary tract, as well as adenocarcinomas which includemalignancies such as most colon cancers, renal-cell carcinoma, prostatecancer and/or testicular tumors, non-small cell carcinoma of the lung,cancer of the small intestine and cancer of the esophagus.

In some embodiments, the methods described herein are used for treatingor diagnosing a carcinoma in a subject. The term “carcinoma” is artrecognized and refers to malignancies of epithelial or endocrine tissuesincluding respiratory system carcinomas, gastrointestinal systemcarcinomas, genitourinary system carcinomas, testicular carcinomas,breast carcinomas, prostatic carcinomas, endocrine system carcinomas,and melanomas. In some embodiments, the cancer is renal carcinoma ormelanoma. Exemplary carcinomas include those forming from tissue of thecervix, lung, prostate, breast, head and neck, colon and ovary. The termalso includes carcinosarcomas, e.g., which include malignant tumorscomposed of carcinomatous and sarcomatous tissues. An “adenocarcinoma”refers to a carcinoma derived from glandular tissue or in which thetumor cells form recognizable glandular structures.

The term “sarcoma” is art recognized and refers to malignant tumors ofmesenchymal derivation.

In some embodiments, the cancers that are treated by the methodsdescribed herein are cancers that have increased levels of mTORC1 or anincreased expression or activity of a mTORC1 relative to normal tissuesor to other cancers of the same tissues; methods known in the art anddescribed herein can be used to identify those cancers. In someembodiments, the methods include obtaining a sample comprising cells ofthe cancer, determining the mTORC1 activity in the sample, andadministering a treatment as described herein (e.g., a providedinhibitor of mTORC1). In some embodiments, the cancer is one that isshown herein to have increased levels of mTORC1 activity.

In some embodiments, the present invention provides a method fortreating one or more disorders, diseases, and/or conditions wherein thedisorder, disease, or condition includes, but is not limited to, acellular proliferative disorder.

Cellular Proliferative Disorders

The present invention features methods and compositions for thediagnosis and prognosis of cellular proliferative disorders (e.g.,cancer) and the treatment of these disorders by inhibiting mTORC1activity. Cellular proliferative disorders described herein include,e.g., cancer, obesity, and proliferation-dependent diseases. Suchdisorders may be diagnosed using methods known in the art.

Cancer

Cancers include, without limitation, leukemias (e.g., acute leukemia,acute lymphocytic leukemia, acute myelocytic leukemia, acutemyeloblastic leukemia, acute promyelocytic leukemia, acutemyelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin'sdisease or non-Hodgkin's disease), Waldenstrom's macroglobulinemia,multiple myeloma, heavy chain disease, and solid tumors such as sarcomasand carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma,meningioma, melanoma, neuroblastoma, and retinoblastoma). In someembodiments, the cancer is melanoma or breast cancer.

Fibrotic Diseases

Idiopathic Pulmonary Fibrosis (IPF). The PI3K pathway is activated infibrotic foci, the cardinal lesions in IPF. mTOR kinase inhibitorGSK2126458 reduces PI3K pathway signaling and functional responses inIPF-derived lung fibroblasts and mTOR inhibition reduces collagenexpression in models of IPF patients. In the bleomycin model ofpulmonary fibrosis, rapamycin treatment is antifibrotic, and rapamycinalso decreases expression of a-smooth muscle actin and fibronectin byfibroblasts in vitro.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat idiopathic pulmonary fibrosis (IPF) (see Mercer, P. F. et al.,Thorax., 71(8): 701-11 (2016); Patel, A. S., et al., PLoS One, 7(7):e41394 (2012)) Accordingly, in some embodiments, the present inventionprovides a method of treating idiopathic pulmonary fibrosis (IPF), in apatient in need thereof, comprising the step of administering to saidpatient a provided compound or pharmaceutically acceptable salt thereof.

Kidney Fibrosis. mTORC1 is activated in myofibroblasts, a majorpathogenic cell type in kidney fibrosis. Inhibition of mTOR withrapamycin in a murine model of kidney fibrosis (UUO), attenuatedexpression of markers of fibrosis and tubulointerstitial damage.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat kidney fibrosis (see Jiang, L., et al., J Am Soc Nephrol, 24(7):1114-26 (2013); Wu, M. J. et al., Kidney International, 69(11): 2029-36(2006); Chen, G. et al., PLoS One, 7(3): e33626 (2012); Liu, C. F. etal., Clin Invest Med, 37(34): E142-53 (2014)). Accordingly, in someembodiments, the present invention provides a method of treating kidneyfibrosis, in a patient in need thereof, comprising the step ofadministering to said patient a provided compound or pharmaceuticallyacceptable salt thereof.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat scleroderma (see Mitra, A., et al., J Invest Dermatol. 135(11):2873-6 (2015)). Accordingly, in some embodiments, the present inventionprovides a method of treating scleroderma, in a patient in need thereof,comprising the step of administering to said patient a provided compoundor pharmaceutically acceptable salt thereof.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat hypertrophic scarring and keloid disease (see Syed, F., et al., AmJ Pathol. 181(5): 1642-58 (2012)). Accordingly, in some embodiments, thepresent invention provides a method of treating hypertrophic scarringand keloid disease, in a patient in need thereof, comprising the step ofadministering to said patient a provided compound or pharmaceuticallyacceptable salt thereof.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat cardiac fibrosis (see Yano, T., et al., J Mol Cell Cardiol. 91:6-9 (2016)). Accordingly, in some embodiments, the present inventionprovides a method of treating cardiac fibrosis, in a patient in needthereof, comprising the step of administering to said patient a providedcompound or pharmaceutically acceptable salt thereof.

Other Proliferative Diseases

Other proliferative diseases include, e.g., obesity, benign prostatichyperplasia, psoriasis, abnormal keratinization, lymphoproliferativedisorders (e.g., a disorder in which there is abnormal proliferation ofcells of the lymphatic system), chronic rheumatoid arthritis,arteriosclerosis, restenosis, and diabetic retinopathy. Proliferativediseases that are hereby incorporated by reference include thosedescribed in U.S. Pat. Nos. 5,639,600 and 7,087,648.

Other Disorders

Other disorders include lysosomal storage diseases, including, but notlimited to, Pompe disease, Gaucher disease, mucopolysaccharidosis,multiple sulfatase deficiency; neurodegenerative diseases such asParkinson's disease, Alzheimer's disease, Huntington's disease,alphal-anti-trypsin deficiency, and spinal bulbar muscular atrophy.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat asthma (see Hua, W., et al., Respirology, 20(7): 1055-65 (2015)).Accordingly, in some embodiments, the present invention provides amethod of treating asthma, in a patient in need thereof, comprising thestep of administering to said patient a provided compound orpharmaceutically acceptable salt thereof.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat a lysosomal storage disease (see Sardiello, M., Annals of the NewYork Academy of Sciences, 1371(1): 3-14 (2016); Awad, O., et al., HumMol Genet. 24(20): 5775-88 (2015); Spampanato, C., et al., EMBO MolMed., 5(5): 691-706 (2013); Medina, D. L., et al., Dev Cell., 21(3):421-30 (2011)). Accordingly, in some embodiments, the present inventionprovides a method of treating a lysosomal storage disease, in a patientin need thereof, comprising the step of administering to said patient aprovided compound or pharmaceutically acceptable salt thereof.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat Parkinson's disease (see Decressac, M., et al., Proc Natl Acad SciUSA., 110(19):E1817-26 (2013)). Accordingly, in some embodiments, thepresent invention provides a method of treating Parkinson's disease, ina patient in need thereof, comprising the step of administering to saidpatient a provided compound or pharmaceutically acceptable salt thereof.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat Alzheimer's disease (see Polito, V. A., et al., EMBO Mol Med.6(9):1142-60 (2014)). Accordingly, in some embodiments, the presentinvention provides a method of treating Alzheimer's disease, in apatient in need thereof, comprising the step of administering to saidpatient a provided compound or pharmaceutically acceptable salt thereof.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat Huntington's disease (see Tsunemi, T., et al., Sci Transl Med.,4(142): 142ra97 (2012)). Accordingly, in some embodiments, the presentinvention provides a method of treating Huntington's disease, in apatient in need thereof, comprising the step of administering to saidpatient a provided compound or pharmaceutically acceptable salt thereof.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat alpha-1-anti-trypsin deficiency (see Pastore, N. et al., EMBO MolMed., 5(3): 397-412 (2013)). Accordingly, in some embodiments, thepresent invention provides a method of treating alphal-anti-trypsindeficiency, in a patient in need thereof, comprising the step ofadministering to said patient a provided compound or pharmaceuticallyacceptable salt thereof.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat spinal bulbar muscular atrophy (see Cortes, C. J., et al., NatNeurosci., 17(9): 1180-9 (2014)). Accordingly, in some embodiments, thepresent invention provides a method of treating spinal bulbar muscularatrophy, in a patient in need thereof, comprising the step ofadministering to said patient a provided compound or pharmaceuticallyacceptable salt thereof.

In some embodiments, a compound of the present invention binds to FKBP12to form a complex. In some embodiments, the complex between a compoundof the present invention and FKBP12 interacts with the FK506-rapamycinbinding domain of mTOR.

In some embodiments, a compound of the present invention binds FKBP12and interferes with protein-protein interaction between FRAP and FKBP12.In some embodiments, the R¹ group of a compound of the present inventioninteracts with both FRAP and FKBP12.

The present invention provides compounds that are inhibitors of mTORC1activity and were shown to selectively inhibit mTORC1 over mTORC2 asmeasured by pS6K inhibition (a measure of mTORC1 activity) and pAKTactivation (a measure of mTORC2 activity). In some embodiments, aprovided compound inhibits mTORC1 selectively over mTORC2. In someembodiments, a provided compound does not measurably inhibit mTORC2. Insome embodiments, a provided compound has a pAKT activation IC₅₀ of >10μM. In some embodiments, a provided compound inhibits mTORC1with >10-fold selectivity over mTORC2. In some embodiments, a providedcompound inhibits mTORC1 with >20-fold selectivity over mTORC2. In someembodiments, a provided compound inhibits mTORC1 with >50-foldselectivity over mTORC2. In some embodiments, a provided compoundinhibits mTORC1 with >100-fold selectivity over mTORC2. In someembodiments, a provided compound inhibits mTORC1 with >150-foldselectivity over mTORC2. In some embodiments, a provided compoundinhibits mTORC1 with >200-fold selectivity over mTORC2. In someembodiments, a provided compound inhibits mTORC1 with >500-foldselectivity over mTORC2. In some embodiments, a provided compoundinhibits mTORC1 with >1,000-fold selectivity over mTORC2.

In some embodiments, a provided compound inhibits mTORC1 selectivelyover mTORC2 after chronic treatment or exposure. In some embodiments, aprovided compound inhibits mTORC1 selectively over mTORC2 after about 24hours of treatment or exposure. In some embodiments, a provided compoundinhibits mTORC1 selectively over mTORC2 after about 36 hours oftreatment or exposure. In some embodiments, a provided compound inhibitsmTORC1 selectively over mTORC2 after about 48 hours of treatment orexposure. In some embodiments, a provided compound inhibits mTORC1selectively over mTORC2 after about 72 hours of treatment or exposure.In some embodiments, a provided compound inhibits mTORC1 selectivelyover mTORC2 after about 96 hours of treatment or exposure. In someembodiments, a provided compound inhibits mTORC1 selectively over mTORC2after about 120 hours of treatment or exposure. In some embodiments, aprovided compound inhibits mTORC1 selectively over mTORC2 after about144 hours of treatment or exposure. In some embodiments, a providedcompound inhibits mTORC1 selectively over mTORC2 after about one week oftreatment or exposure. In some embodiments, a provided compound inhibitsmTORC1 selectively over mTORC2 after more than about one week oftreatment or exposure.

In some embodiments, a provided compound is less immunosuppressive thanexisting rapalogs. In some embodiments, a provided compound is lessimmunosuppressive than rapamycin. In some embodiments, a providedcompound is less immunosuppressive than everolimus. In some embodiments,a provided compound is less immunosuppressive than temsirolimus. In someembodiments, a provided compound is less immunosuppressive thanridaforolimus. In some embodiments, a provided compound is lessimmunosuppressive than umirolimus.

In some embodiments, a provided compound suppresses interferon gamma(IFN-γ) production less than rapalogs. In some embodiments, a providedcompound suppresses IFN-γ production less than rapamycin. In someembodiments, a provided compound suppresses IFN-γ production less thaneverolimus. In some embodiments, a provided compound suppresses IFN-γproduction less than temsirolimus. In some embodiments, a providedcompound suppresses IFN-γ production less than ridaforolimus. In someembodiments, a provided compound suppresses IFN-γ production less thanumirolimus.

In some embodiments, a provided compound decreases the expression offibrosis biomarkers in tissue that has been damaged. In someembodiments, a provided compound decreases the expression of collagen I(COL1A2) in tissue that has been damaged. In some embodiments, aprovided compound decreases the expression of collagen III (COL3A1) intissue that has been damaged. In some embodiments, a provided compounddecreases the expression of fibronectin (FN1) in tissue that has beendamaged.

In some embodiments, a provided compound decreases the propensity ofimmune cells from infiltrating damaged tissue. In some embodiments, aprovided compound decreases the propensity of macrophage cells frominfiltrating damaged tissue.

In some embodiments, a provided compound induces less glucose tolerancethan rapalogs. In some embodiments, a provided compound induces lessglucose tolerance than rapamycin. In some embodiments, a providedcompound induces less glucose tolerance than everolimus. In someembodiments, a provided compound induces less glucose tolerance thantemsirolimus. In some embodiments, a provided compound induces lessglucose tolerance than ridaforolimus. In some embodiments, a providedcompound induces less glucose tolerance than umirolimus. In someembodiments, a provided compound does not induce glucose tolerancesignificantly more than a placebo or vehicle alone.

Accordingly, in some embodiments, the present invention provides amethod of treating a disorder associate with mTORC1 comprisingadministering to patient a compound that inhibits mTORC1 wherein saidcompound does not inhibit mTORC2. Such compounds may be employed forindications where rapamycin and rapalogs demonstrated a benefit eitherin animal models or in a human disease setting. Such indicationsinclude:

Treatment of Metabolic Disease (Obesity and Insulin Resistance in Type 2Diabetes). Inhibition of mTORC1 pathway leads to extension of life spanin yeast, fly and mouse, and caloric restriction improves longevity andinsulin sensitivity. The underlying mechanism has been proposed tofunction by regulation of mTORC1 activation. Rapamycin-induced insulinresistance has been shown to be mediated by inhibition of mTORC2 andselective mTORC1 inhibitor is predicted to improve insulin sensitivityand glucose homeostasis.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat metabolic disease (obesity and insulin resistance in type 2diabetes) (see Yu, Z., et al., J Gerontol A Biol Sci Med Sci, 70(4),410-20 (2015); Fok, W. C., et al., Aging Cell 13 (2): 311-9 (2014);Shum, M., et al., Diabetologia, 59(3):592-603 (2016); Lamming, D. W., etal., Science 335(6076): 1638-43 (2012)). Accordingly, in someembodiments, the present invention provides a method of treatingmetabolic disease (obesity and insulin resistance in type 2 diabetes),in a patient in need thereof, comprising the step of administering tosaid patient a provided compound or pharmaceutically acceptable saltthereof.

Neurofibromatosis. Neurofibromatosis type 1 (NF1) is caused by mutationsin the NF1 gene. Its protein product, neurofibromin, functions as atumor suppressor and ultimately produces constitutive upregulation ofmTOR. mTOR inhibitors have been shown to reduce tumor size and induceanti-proliferative effect in NF1-associated plexiform neurofibroma.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat neurofibromatosis (see Franz, D. N., et al., Curr Neurol NeurosciRep., 12(3): 294-301 (2012); Varin, J., et al., Oncotarget., 7: 35753-67(2016)). Accordingly, in some embodiments, the present inventionprovides a method of treating neurofibromatosis, in a patient in needthereof, comprising the step of administering to said patient a providedcompound or pharmaceutically acceptable salt thereof.

Cardiomyopathy and skeletal muscle dystrophy, Emery-Dreifuss musculardystrophy model (LMNA^(−/−)). Mutations in LMNA result in several humandiseases including limb-girdle muscular dystrophy (LGMD1B),Emery-Dreifuss muscular dystrophy (EDMD⅔), dilated cardiomyopathy (DCM)and conduction-system disease (CMD1A), lipodystrophy,Charcot-Marie-Tooth disease, and Hutchinson-Gilford progeria syndrome(HGPS). Lmna^(−/−) mice have elevated mTORC1 activity and short-termtreatment with rapamycin in Lmna^(−/−) mice results in reduced mTORC1signaling, improved cardiac and skeletal muscle function and enhancedsurvival by ˜50%.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat cardiomyopathy and skeletal muscle dystrophy (see Ramos, F., etal., Sci Transl Med., 4(144): 144ra103 (2012); Bonne, G. & Quijano-Roy,S., Handb Clin Neurol., 113: 1367-76 (2013)). Accordingly, in someembodiments, the present invention provides a method of treatingcardiomyopathy and skeletal muscle dystrophy, in a patient in needthereof, comprising the step of administering to said patient a providedcompound or pharmaceutically acceptable salt thereof.

Leigh syndrome. Ndufs4 knockout (KO) mice are used as a model of Leighsyndrome and exhibit hyperactivation of mTORC1 and metabolic defects.Treatment of Ndufs4 KO mice with rapamycin extended lifespan, improvemetabolic and neurological defect associated with this disease.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat Leigh syndrome (see Johnson, S.C., et al., Science, 342(6165):1524-8 (2013)). Accordingly, in some embodiments, the present inventionprovides a method of treating Leigh syndrome, in a patient in needthereof, comprising the step of administering to said patient a providedcompound or pharmaceutically acceptable salt thereof.

Oncology. Inhibition of mTOR with rapalogs has been shown to haveantitumor activity in murine cancer models and in cancer patients.Examples of sensitive cancer types include, but are not limited to,hepatocellular carcinoma, breast cancers, mantle cell lymphomas, lungcarcinoma, tuberous sclerosis and lymphangioleiomyomatosis.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat cancer and oncologic disorders (see Ilagan, E. & manning, B. D.,Trends Cancer, 2(5): 241-51 (2016)). Accordingly, in some embodiments,the present invention provides a method of treating cancer and oncologicdisorders, in a patient in need thereof, comprising the step ofadministering to said patient a provided compound or pharmaceuticallyacceptable salt thereof.

Non-alcoholic steatohepatitis (NASH). The present invention providesinhibitors that induce autophagy to clear degraded cytoplasmic proteins,and NASH disease is characterized by lipid deposits, inflammation andfibrosis in the liver. The inhibition of mTORC1 pathway induce autophagyand down regulate SREBP-1 to decrease lipid biosynthesis to reduce lipidstorage.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat non-alcoholic steatohepatitis (NASH) (see Puri, P. & Chandra, A.,J Clin Exp Hepatol, 4(1): 51-9 (2014)). Accordingly, in someembodiments, the present invention provides a method of treatingnon-alcoholic steatohepatitis (NASH), in a patient in need thereof,comprising the step of administering to said patient a provided compoundor pharmaceutically acceptable salt thereof.

Tuberous sclerosis (TSC) and lymphangioleiomyomatosis (LAM). Failure inthe regulation of mTOR is critical to the pathogenesis of the inheriteddisorder tuberous sclerosis complex (TSC) and the related lung disease,lymphangioleiomyomatosis (LAM). Both diseases are caused by mutations ofTSC1 or TSC2 leading to inappropriate activity of signaling downstreamof mTORC1. TSC patients develop nonmalignant tumors in many organs,including the brain, while LAM patients, mostly women, accumulateabnormal, muscle-like cells in certain organs or tissues, especially thelungs, lymph nodes, and kidneys. The rapalogs, everolimus and sirolimus,are currently approved for the treatment of both TSC and LAM,respectively, by the U.S. FDA.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat tuberous sclerosis and lymphangioleiomyomatosis (see Wander, S.A., et al., J. Clin. Invest., 121(4): 1231-41 (2011); Taveira-DaSilva,A. M. & Moss, J., J. Clin Epidemiol., 7: 249-57 (2015)). Accordingly, insome embodiments, the present invention provides a method of treatingtuberous sclerosis and lymphangioleiomyomatosis, in a patient in needthereof, comprising the step of administering to said patient a providedcompound or pharmaceutically acceptable salt thereof.

Senescence and diseases of aging. Rapamycin suppresses the mammalianTORC1 complex, which regulates translation, and extends lifespan indiverse species, including mice. Rapamycin was shown to inhibit thepro-inflammatory phenotype of senescent cells. As senescent cellsaccumulate with age, the senescence-associated secretory phenotype(SASP) can disrupt tissues and contribute to age-related pathologies,including cancer. Inhibition of mTOR suppressed the secretion ofinflammatory cytokines by senescent cells. Rapamycin reduced cytokinelevels including IL6 and suppressed translation of the membrane-boundcytokine IL1A. Reduced IL1A diminishes NF-κB transcriptional activity,which controls the SASP. Thus, mTORC1 inhibitors might ameliorateage-related pathologies, including late-life cancer, by suppressingsenescence-associated inflammation.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat senescence and diseases of aging (see Laberge, R. M., et al.,Nature Cell Biology, 17(8): 1049-61 (2015); Nacarelli, T., et al., FreeRadic Biol Med., 95: 133-54 (2016)). Accordingly, in some embodiments,the present invention provides a method of treating senescence anddiseases of aging, in a patient in need thereof, comprising the step ofadministering to said patient a provided compound or pharmaceuticallyacceptable salt thereof.

Diabetic nephropathy and kidney-related complications of type 1 diabetesand type 2 diabetes. Diabetic nephropathy is a kidney complication oftype-1 and type-2 diabetes, affecting up to nearly 40% of people withdiabetes. High levels of glucose force the kidneys work excessively tofilter blood, resulting in kidney damage. Studies suggest that the mTORpathway is highly activated in patients with diabetic neuropathy and mayplay a role in the pathological changes and renal dysfunction due tochronic high glucose. Further, mTOR inhibition may attenuatehyperinsulinemia.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat diabetic nephropathy or kidney-related complications of type 1diabetes and type 2 diabetes (see Mori, H., et al., Biochem. Res.Commun. 384(4): 471-5 (2009)). Accordingly, in some embodiments, thepresent invention provides a method of treating diabetic nephropathy orkidney-related complications of type 1 diabetes and type 2 diabetes in apatient in need thereof, comprising the step of administering to saidpatient a provided compound or pharmaceutically acceptable salt thereof.

Polycystic kidney disease. Polycystic kidney disease (PKD) ischaracterized by the development and accumulation of destructive kidneycysts that eventually result in kidney failure. PKD may be autosomaldominant (ADPKD) or recessive (ARPKD). Dysfunctional mTOR signalingpathway has been observed in ADPKD and ARPKD. Thus, normalization of themTORC1 pathway may ameliorate the development of cysts and progressionof the disease.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat PKD (see Torres, V. E., et al., Clin. J. Am. Soc. Nephrol. 5(7):1312-29 (2010)). Accordingly, in some embodiments, the present inventionprovides a method of treating PKD in a patient in need thereof,comprising the step of administering to said patient a provided compoundor pharmaceutically acceptable salt thereof. In some embodiments, PKD isautosomal dominate. In some embodiments, PKD is autosomal recessive.

Focal Segmental Glomerulosclerosis (FSGS) and other diseases associatedwith sclerosis of the kidney. FSGS is the most common primary glomerulardisorder causing end-stage renal disease (ESRD) in the United States. Asthe disease progresses there is a mismatch of podocyte cells in Bowman'scapsule and the surface area of the glomerular basement membrane theycover. Studies have shown that podocyte size control is regulated bymTOR and that mTOR activation contributes to disease progression.Further, constitutive mTORC1 activation has been shown to causeFSGS-like lesions in mouse knockdown experiments. Thus, mTORC1inhibition might ameliorate (FSGS) or other diseases associated withsclerosis of the kidney by normalizing or increasing autophagicactivity.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat FSGS or other diseases associated with sclerosis of the kidney(see Zschiedrich, S. et al., J. Am. Soc. Nephrol. 28(7): 2144-57(2017)). Accordingly, in some embodiments, the present inventionprovides a method of treating FSGS or other diseases associated withsclerosis of the kidney in a patient in need thereof, comprising thestep of administering to said patient a provided compound orpharmaceutically acceptable salt thereof.

Age-Related Macular Degeneration. Age-related macular degeneration (AMD)is a leading cause of blindness characterized by the death ofphotoreceptors in the macula. Possible mechanisms of AMD progressioninclude oxidative stress leading to deposits of proteins anddysfunctional organelles, leading to retinal pigment epitheliumhypertrophy, dedifferentiation, and eventual atrophy. mTOR is implicatedin the dedifferentiation of the retinal pigment epithelium. Thus, mTORC1inhibition may ameliorate AMD by blocking hypertrophy anddedifferentiation.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat age-related macular degeneration (see Kolosova, N. G., et al., Am.J. Path. 181(2): 472-7 (2012) and Zhen, C. & Vollrath, D., Aging 3(4):346-47 (2011)). Accordingly, in some embodiments, the present inventionprovides a method of treating age-related macular degeneration in apatient in need thereof, comprising the step of administering to saidpatient a provided compound or pharmaceutically acceptable salt thereof.

Diabetic Macular Edema. Diabetic macular edema (DME) is a leading causeof blindness in persons with diabetes, affecting approximately 35% ofpeople with diabetes. Studies suggest that the pathogenesis of DME is aninflammatory disease involving various cytokines and chemokines. Chronicinflammatory and oxidative stress may contribute to the progression ofDME. Thus, inhibition of mTORC1 may ameliorate DME symptoms andprogression by decreasing the inflammatory response.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat DME (see Okamoto, T., et al., PLOS ONE, (11)(1): e0146517,https://doi.org/10.1371/journal.pone.0146517 (2016)). Accordingly, insome embodiments, the present invention provides a method of treatingDME in a patient in need thereof, comprising the step of administeringto said patient a provided compound or pharmaceutically acceptable saltthereof.

Diabetic retinopathy. Diabetic retinopathy (DR) is a common eye diseaseaccounting for 5% of blindness in adults and is associated with chronichyperglycemia and defects of insulin signaling pathways. DR patientssuffer persistent injury to retinal blood vessels and neurons byinflammation, reactive oxygen species and endoplasmic reticulum stresscaused by chronic hyperglycemia. Significantly, rapamycin has been shownto block the action of insulin-induced hypoxia-inducible factor-1(HIF-1) and retinal cell senescence, and induces autophagy, and could bebeneficial in promoting apoptosis of nascent blood vessels andpreventing angiogenesis. Thus, inhibition of mTORC1 may ameliorate DRsymptoms and progression by decreasing inflammation and inhibitingpathogenic signaling pathways.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat DR (see Di Rosa, M., et al., Curr. Neuropharmacol. 14(8): 810-25(2016)). Accordingly, in some embodiments, the present inventionprovides a method of treating DR in a patient in need thereof,comprising the step of administering to said patient a provided compoundor pharmaceutically acceptable salt thereof.

Glaucoma. Glaucoma is a common optic neuropathy associated with agingand elevated intraocular pressure, and is the leading cause ofirreversible blindness. Studies suggest that mTOR dependentdysregulation of autophagocytosis may be a factor in the progression ofthe disease. Thus, inhibition of mTORC1 may slow the progression orameliorate glaucoma by normalizing or increasing autophagy.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat glaucoma (see Porter, K., et al., Biochim. Biophys. Acta. 1852(3):379-85 (2014)). Accordingly, in some embodiments, the present inventionprovides a method of treating glaucoma in a patient in need thereof,comprising the step of administering to said patient a provided compoundor pharmaceutically acceptable salt thereof.

Restoring immune function. mTORC1 inhibition has been shown to reducethe expression of programmed death-1 (PD-1) receptor in CD4⁺ and CD8⁺ Tlymphocytes, promoting T-cell signaling. Thus, mTORC1 inhibition mayrestore immune function by improving the adaptive immune response.

In some embodiments, the method of inhibiting mTORC1 activity is used torestore immune function (see Mannick, J. B., et al., Sci. Trans. Med.6(268): ppra179 (2014)). Accordingly, in some embodiments, the presentinvention provides a method of restoring immune function in a patient inneed thereof, comprising the step of administering to said patient aprovided compound or pharmaceutically acceptable salt thereof.

Treatment of respiratory and/or urinary tract infections. mTORC1inhibition may reduce infections by upregulation of antiviral geneexpression and response. Thus, mTORC1 inhibition may enhance the abilityof a patient's immune system to defend against respiratory and/orurinary tract infections.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat respiratory and/or urinary tract infections. (see Mannick, J. B.,et al., Sci. Trans. Med. 10(449): eaaq1564 (2018)). Accordingly, in someembodiments, the present invention provides a method of restoring immunefunction in a patient in need thereof, comprising the step ofadministering to said patient a provided compound or pharmaceuticallyacceptable salt thereof.

Heart failure. mTORC1 activity is essential for cardiac hypertrophy inresponse to stress but can lead to cardiac derangements as a result ofcardiac remodeling following infarction. Inhibition of mTORC1 reducescardiac remodeling and heart failure in response to pressure overload.Thus, inhibition of mTORC1 may decrease heart failure in patients whohave suffered damage to the myocardium.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat heart failure (see Sciarretta, S. et al., Circ. Res. 122(3):489-505 (2018)). Accordingly, in some embodiments, the present inventionprovides a method of treating heart failure in a patient in needthereof, comprising the step of administering to said patient a providedcompound or pharmaceutically acceptable salt thereof.

Osteoarthritis. Osteoarthritis (OA) is a chronic degenerative diseaseresulting in loss of cartilage and joint inflammation. mTOR may play asignificant role in collagen homeostasis and turnover and remodeling ofcartilage. Thus, inhibition of mTORC1 may slow the progression orameliorate osteoarthritis symptoms by normalizing cartilage turnover.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat osteoarthritis (see Pal, B., et al., Drugs R&D, 15(1): 27-36(2017))). Accordingly, in some embodiments, the present inventionprovides a method of treating osteoarthritis in a patient in needthereof, comprising the step of administering to said patient a providedcompound or pharmaceutically acceptable salt thereof.

Pulmonary arterial hypertension. Pulmonary arterial hypertension (PAH)is a progressive, fatal disease associated with increases pulmonaryvascular resistance. Pulmonary arterial smooth muscle cell proliferationand migration are implicated in the progressing of arterial wallthickening, exacerbating vasoconstriction. Thus, inhibition of mTORC1may alleviate PAH by reducing vascular remodeling.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat PAH (see Ma, X., et al., Interact. Cardiovasc. Thorac. Surg.25(2): 206-11 (2017)). Accordingly, in some embodiments, the presentinvention provides a method of treating PAH is a patient in needthereof, comprising the step of administering to said patient a providedcompound or pharmaceutically acceptable salt thereof.

Chronic Obstructive Pulmonary Disease. Reduced autophagy results in theaccumulation of proteins and other cellular materials that acceleratecellular senescence in patients with chronic obstructive pulmonarydisease (COPD). Thus, inhibition of mTORC1 may slow the progression orameliorate COPD symptoms by normalizing or increasing autophagy.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat COPD (see Fujii, S., et al., Oncoimmunology 1(5): 630-41 (2012)).Accordingly, in some embodiments, the present invention provides amethod of treating COPD in a patient in need thereof, comprising thestep of administering to said patient a provided compound orpharmaceutically acceptable salt thereof.

Additional therapeutic indications where mTORC1 inhibition may bebeneficial are: cardiovascular disease (acute coronary syndrome),coronary occlusions with eluting stents, polycystic kidney disease, andkidney disease associated with cyst formation or cystogenesis),neurofibromatosis, epilepsy assoc. with TSC1 and/or TSC2 mutations,polycystic liver, pachyonychia congenital, fragile x syndrome, Friedrichataxia, Peutz-Jeghers syndrome, eye disease including neovascularage-related macular degeneration, uveitis, diabetic macular edema,fibroblast growth including pulmonary fibrosis, renalinsufficiency/fibrosis, metabolic syndrome, diseases of the immunesystem including immune senescence, lupus nephritis, chronic immunethrombocytopenia, multiple sclerosis, cancer including lymphoma, tumorsassociated with TSC½ mutations, angiomyolipoma assoc. with TSC½mutations, breast cancer, hepatocellular cancer, leukemia, glioma,adenoid cystic carcinoma, senescence, autism, and vascular rheumatoidarthritis.

In some embodiments, the method of inhibiting mTORC1 activity is used totreat cardiovascular disease (acute coronary syndrome), coronaryocclusions with eluting stents, polycystic kidney disease,neurofibromatosis, epilepsy assoc. with TSC1 and/or TSC2 mutations,polycystic liver, pachyonychia congenital, fragile x syndrome, Friedrichataxia, Peutz-Jeghers syndrome, eye disease including neovascularage-related macular degeneration, uveitis, diabetic macular edema,fibroblast growth including pulmonary fibrosis, renalinsufficiency/fibrosis, metabolic syndrome, diseases of the immunesystem including immune senescence, lupus nephritis, chronic immunethrombocytopenia, multiple sclerosis, cancer including lymphoma, tumorsassociated with TSC½ mutations, angiomyolipoma associated with TSC½mutations, breast cancer, hepatocellular cancer, leukemia, glioma,adenoid cystic carcinoma, senescence, autism, and vascular rheumatoidarthritis.

Accordingly, in some embodiments, the present invention provides amethod of treating cardiovascular disease (acute coronary syndrome),coronary occlusions with eluting stents, polycystic kidney disease,neurofibromatosis, epilepsy assoc. with TSC1 and/or TSC2 mutations,polycystic liver, pachyonychia congenital, fragile x syndrome, Friedrichataxia, Peutz-Jeghers syndrome, eye disease including neovascularage-related macular degeneration, uveitis, diabetic macular edema,fibroblast growth including pulmonary fibrosis, renalinsufficiency/fibrosis, metabolic syndrome, diseases of the immunesystem including immune senescence, lupus nephritis, chronic immunethrombocytopenia, multiple sclerosis, cancer including lymphoma, tumorsassociated with TSC½ mutations, angiomyolipoma assoc. with TSC½mutations, breast cancer, hepatocellular cancer, leukemia, glioma,adenoid cystic carcinoma, senescence, autism, and vascular rheumatoidarthritis, in a patient in need thereof, comprising the step ofadministering to said patient a provided compound or pharmaceuticallyacceptable salt thereof.

Pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated. Incertain embodiments, the compounds of the invention may be administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium priorto use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms can be made by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

The term “biological sample”, as used herein, includes, withoutlimitation, cell cultures or extracts thereof; biopsied materialobtained from a mammal or extracts thereof; and blood, saliva, urine,feces, semen, tears, or other body fluids or extracts thereof.

In other embodiments, the present invention provides a method fortreating a disorder mediated by mTORC1 in a patient in need thereof,comprising the step of administering to said patient a compoundaccording to the present invention or pharmaceutically acceptablecomposition thereof. Such disorders are described in detail herein.

Depending upon the particular condition, or disease, to be treated,additional therapeutic agents that are normally administered to treatthat condition, may also be present in the compositions of thisinvention. As used herein, additional therapeutic agents that arenormally administered to treat a particular disease, or condition, areknown as “appropriate for the disease, or condition, being treated.”

A compound of the current invention may also be used to advantage incombination with other antiproliferative compounds. Suchantiproliferative compounds include, but are not limited to aromataseinhibitors; antiestrogens; topoisomerase I inhibitors; topoisomerase IIinhibitors; microtubule active compounds; alkylating compounds; histonedeacetylase inhibitors; compounds which induce cell differentiationprocesses; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors;antineoplastic antimetabolites; platin compounds; compoundstargeting/decreasing a protein or lipid kinase activity and furtheranti-angiogenic compounds; compounds which target, decrease or inhibitthe activity of a protein or lipid phosphatase; gonadorelin agonists;anti-androgens; methionine aminopeptidase inhibitors; matrixmetalloproteinase inhibitors; bisphosphonates; biological responsemodifiers; antiproliferative antibodies; heparanase inhibitors;inhibitors of Ras oncogenic isoforms; telomerase inhibitors; proteasomeinhibitors; compounds used in the treatment of hematologic malignancies;compounds which target, decrease or inhibit the activity of Flt-3; Hsp90inhibitors such as 17-AAG (17-allylaminogeldanamycin, NSC330507),17-DMAG (17-dimethylaminoethylamino-17-demethoxy-geldanamycin,NSC707545), IPI-504, CNF1010, CNF2024, CNF1010 from ConformaTherapeutics; temozolomide (Temodal®); kinesin spindle proteininhibitors, such as SB715992 or SB743921 from GlaxoSmithKline, orpentamidine/chlorpromazine from CombinatoRx; MEK inhibitors such asARRY142886 from Array BioPharma, AZD6244 from AstraZeneca, PD181461 fromPfizer and leucovorin. The term “aromatase inhibitor” as used hereinrelates to a compound which inhibits estrogen production, for instance,the conversion of the substrates androstenedione and testosterone toestrone and estradiol, respectively. The term includes, but is notlimited to steroids, especially atamestane, exemestane and formestaneand, in particular, non-steroids, especially aminoglutethimide,roglethimide, pyridoglutethimide, trilostane, testolactone,ketokonazole, vorozole, fadrozole, anastrozole and letrozole. Exemestaneis marketed under the trade name Aromasin™. Formestane is marketed underthe trade name Lentaron™. Fadrozole is marketed under the trade nameAfema™. Anastrozole is marketed under the trade name Arimidex™.Letrozole is marketed under the trade names Femara™ or Femar™.Aminoglutethimide is marketed under the trade name Orimeten™. Acombination of the invention comprising a chemotherapeutic agent whichis an aromatase inhibitor is particularly useful for the treatment ofhormone receptor positive tumors, such as breast tumors.

The term “antiestrogen” as used herein relates to a compound whichantagonizes the effect of estrogens at the estrogen receptor level. Theterm includes, but is not limited to tamoxifen, fulvestrant, raloxifeneand raloxifene hydrochloride. Tamoxifen is marketed under the trade nameNolvadex™. Raloxifene hydrochloride is marketed under the trade nameEvista™. Fulvestrant can be administered under the trade name Faslodex™.A combination of the invention comprising a chemotherapeutic agent whichis an antiestrogen is particularly useful for the treatment of estrogenreceptor positive tumors, such as breast tumors.

The term “anti-androgen” as used herein relates to any substance whichis capable of inhibiting the biological effects of androgenic hormonesand includes, but is not limited to, bicalutamide (Casodex™). The term“gonadorelin agonist” as used herein includes, but is not limited toabarelix, goserelin and goserelin acetate. Goserelin can be administeredunder the trade name Zoladex™.

The term “topoisomerase I inhibitor” as used herein includes, but is notlimited to topotecan, gimatecan, irinotecan, camptothecian and itsanalogues, 9-nitrocamptothecin and the macromolecular camptothecinconjugate PNU-166148. Irinotecan can be administered, e.g., in the formas it is marketed, e.g. under the trademark Camptosar™. Topotecan ismarketed under the trade name Hycamptin™.

The term “topoisomerase II inhibitor” as used herein includes, but isnot limited to the anthracyclines such as doxorubicin (includingliposomal formulation, such as Caelyx™) daunorubicin, epirubicin,idarubicin and nemorubicin, the anthraquinones mitoxantrone andlosoxantrone, and the podophillotoxines etoposide and teniposide.Etoposide is marketed under the trade name Etopophos™. Teniposide ismarketed under the trade name VM 26-Bristol Doxorubicin is marketedunder the trade name Acriblastin™ or Adriamycin™. Epirubicin is marketedunder the trade name Farmorubicin™. Idarubicin is marketed. under thetrade name Zavedos™. Mitoxantrone is marketed under the trade nameNovantron.

The term “microtubule active agent” relates to microtubule stabilizing,microtubule destabilizing compounds and microtublin polymerizationinhibitors including, but not limited to taxanes, such as paclitaxel anddocetaxel; vinca alkaloids, such as vinblastine or vinblastine sulfate,vincristine or vincristine sulfate, and vinorelbine; discodermolides;cochicine and epothilones and derivatives thereof. Paclitaxel ismarketed under the trade name Taxol™. Docetaxel is marketed under thetrade name Taxotere™. Vinblastine sulfate is marketed under the tradename Vinblastin R.P™. Vincristine sulfate is marketed under the tradename Farmistin™.

The term “alkylating agent” as used herein includes, but is not limitedto, cyclophosphamide, ifosfamide, melphalan or nitrosourea (BCNU orGliadel). Cyclophosphamide is marketed under the trade name Cyclostin™.Ifosfamide is marketed under the trade name Holoxan™.

The term “histone deacetylase inhibitors” or “HDAC inhibitors” relatesto compounds which inhibit the histone deacetylase and which possessantiproliferative activity. This includes, but is not limited to,suberoylanilide hydroxamic acid (SAHA).

The term “antineoplastic antimetabolite” includes, but is not limitedto, 5-fluorouracil or 5-FU, capecitabine, gemcitabine, DNA demethylatingcompounds, such as 5-azacytidine and decitabine, methotrexate andedatrexate, and folic acid antagonists such as pemetrexed. Capecitabineis marketed under the trade name Xeloda™. Gemcitabine is marketed underthe trade name Gemzar™.

The term “platin compound” as used herein includes, but is not limitedto, carboplatin, cis-platin, cisplatinum and oxaliplatin. Carboplatincan be administered, e.g., in the form as it is marketed, e.g., underthe trademark Carboplat™. Oxaliplatin can be administered, e.g., in theform as it is marketed, e.g., under the trademark Eloxatin™.

The term “compounds targeting/decreasing a protein or lipid kinaseactivity; or a protein or lipid phosphatase activity; or furtheranti-angiogenic compounds” as used herein includes, but is not limitedto, protein tyrosine kinase and/or serine and/or threonine kinaseinhibitors or lipid kinase inhibitors, such as a) compounds targeting,decreasing or inhibiting the activity of the platelet-derived growthfactor-receptors (PDGFR), such as compounds which target, decrease orinhibit the activity of PDGFR, especially compounds which inhibit thePDGF receptor, such as an N-phenyl-2-pyrimidine-amine derivative, suchas imatinib, SU101, SU6668 and GFB-111; b) compounds targeting,decreasing or inhibiting the activity of the fibroblast growthfactor-receptors (FGFR); c) compounds targeting, decreasing orinhibiting the activity of the insulin-like growth factor receptor I(IGF-IR), such as compounds which target, decrease or inhibit theactivity of IGF-IR, especially compounds which inhibit the kinaseactivity of IGF-I receptor, or antibodies that target the extracellulardomain of IGF-I receptor or its growth factors; d) compounds targeting,decreasing or inhibiting the activity of the Trk receptor tyrosinekinase family, or ephrin B4 inhibitors; e) compounds targeting,decreasing or inhibiting the activity of the AxI receptor tyrosinekinase family; f) compounds targeting, decreasing or inhibiting theactivity of the Ret receptor tyrosine kinase; g) compounds targeting,decreasing or inhibiting the activity of the Kit/SCFR receptor tyrosinekinase, such as imatinib; h) compounds targeting, decreasing orinhibiting the activity of the C-kit receptor tyrosine kinases, whichare part of the PDGFR family, such as compounds which target, decreaseor inhibit the activity of the c-Kit receptor tyrosine kinase family,especially compounds which inhibit the c-Kit receptor, such as imatinib;i) compounds targeting, decreasing or inhibiting the activity of membersof the c-Abl family, their gene-fusion products (e.g., BCR-Abl kinase)and mutants, such as compounds which target decrease or inhibit theactivity of c-Abl family members and their gene fusion products, such asan N-phenyl-2-pyrimidine-amine derivative, such as imatinib or nilotinib(AMN107); PD180970; AG957; NSC 680410; PD173955 from ParkeDavis; ordasatinib (BMS-354825); j) compounds targeting, decreasing or inhibitingthe activity of members of the protein kinase C (PKC) and Raf family ofserine/threonine kinases, members of the MEK, SRC, JAK/pan-JAK, FAK,PDK1, PKB/Akt, Ras/MAPK, PI3K, SYK, TYK2, BTK and TEC family, and/ormembers of the cyclin-dependent kinase family (CDK) includingstaurosporine derivatives, such as midostaurin; examples of furthercompounds include UCN-01, safingol, BAY 43-9006, Bryostatin 1,Perifosine; llmofosine; RO 318220 and RO 320432; GO 6976; lsis 3521;LY333531/LY379196; isochinoline compounds; FTIs; PD184352 or QAN697 (aPI3K inhibitor) or AT7519 (CDK inhibitor); k) compounds targeting,decreasing or inhibiting the activity of protein-tyrosine kinaseinhibitors, such as compounds which target, decrease or inhibit theactivity of protein-tyrosine kinase inhibitors include imatinib mesylate(Gleevec™) or tyrphostin such as Tyrphostin A23/RG-50810; AG 99;Tyrphostin AG 213; Tyrphostin AG 1748; Tyrphostin AG 490; TyrphostinB44; Tyrphostin B44 (+) enantiomer; Tyrphostin AG 555; AG 494;Tyrphostin AG 556, AG957 and adaphostin(4-{[(2,5-dihydroxyphenyl)methyl]amino}-benzoic acid adamantyl ester;NSC 680410, adaphostin); 1) compounds targeting, decreasing orinhibiting the activity of the epidermal growth factor family ofreceptor tyrosine kinases (EGFR₁ ErbB2, ErbB3, ErbB4 as homo- orheterodimers) and their mutants, such as compounds which target,decrease or inhibit the activity of the epidermal growth factor receptorfamily are especially compounds, proteins or antibodies which inhibitmembers of the EGF receptor tyrosine kinase family, such as EGFreceptor, ErbB2, ErbB3 and ErbB4 or bind to EGF or EGF related ligands,CP 358774, ZD 1839, ZM 105180; trastuzumab (Herceptin™), cetuximab(Erbitux™), Iressa, Tarceva, OSI-774, Cl-1033, EKB-569, GW-2016, E1.1,E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 or E7.6.3, and7H-pyrrolo-[2,3-d]pyrimidine derivatives; m) compounds targeting,decreasing or inhibiting the activity of the c-Met receptor, such ascompounds which target, decrease or inhibit the activity of c-Met,especially compounds which inhibit the kinase activity of c-Metreceptor, or antibodies that target the extracellular domain of c-Met orbind to HGF, n) compounds targeting, decreasing or inhibiting the kinaseactivity of one or more JAK family members (JAK1/JAK2/JAK3/TYK2 and/orpan-JAK), including but not limited to PRT-062070, SB-1578, baricitinib,pacritinib, momelotinib, VX-509, AZD-1480, TG-101348, tofacitinib, andruxolitinib; o) compounds targeting, decreasing or inhibiting the kinaseactivity of PI3 kinase (PI3K) including but not limited to ATU-027,SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474, buparlisib,pictrelisib, PF-4691502, BYL-719, dactolisib, XL-147, XL-765, andidelalisib; and; and q) compounds targeting, decreasing or inhibitingthe signaling effects of hedgehog protein (Hh) or smoothened receptor(SMO) pathways, including but not limited to cyclopamine, vismodegib,itraconazole, erismodegib, and IPI-926 (saridegib).

The term “PI3K inhibitor” as used herein includes, but is not limited tocompounds having inhibitory activity against one or more enzymes in thephosphatidylinositol-3-kinase family, including, but not limited toPI3Kα, PI3Kγ, PI3Kδ, PI3Kβ, PI3K-C2α, PI3K-C2β, PI3K-C2γ, Vps34, p110-α,p110-β, p110-γ, p110-δ, p85-α, p85-β, p55-γ, p150, p101, and p87.Examples of PI3K inhibitors useful in this invention include but are notlimited to ATU-027, SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474,buparlisib, pictrelisib, PF-4691502, BYL-719, dactolisib, XL-147,XL-765, and idelalisib.

The term “Bcl-2 inhibitor” as used herein includes, but is not limitedto compounds having inhibitory activity against B-cell lymphoma 2protein (Bcl-2), including but not limited to ABT-199, ABT-731, ABT-737,apogossypol, Ascenta's pan-Bcl-2 inhibitors, curcumin (and analogsthereof), dual Bcl-2/Bcl-xL inhibitors (InfinityPharmaceuticals/Novartis Pharmaceuticals), Genasense (G3139), HA14-1(and analogs thereof; see WO 2008/118802), navitoclax (and analogsthereof, see U.S. Pat. No. 7,390,799), NH-1 (Shenayng PharmaceuticalUniversity), obatoclax (and analogs thereof, see WO 2004/106328), S-001(Gloria Pharmaceuticals), TW series compounds (Univ. of Michigan), andvenetoclax. In some embodiments the Bcl-2 inhibitor is a small moleculetherapeutic. In some embodiments the Bcl-2 inhibitor is apeptidomimetic.

The term “BTK inhibitor” as used herein includes, but is not limited tocompounds having inhibitory activity against Bruton's Tyrosine Kinase(BTK), including, but not limited to AVL-292 and ibrutinib.

The term “SYK inhibitor” as used herein includes, but is not limited tocompounds having inhibitory activity against spleen tyrosine kinase(SYK), including but not limited to PRT-062070, R-343, R-333, Excellair,PRT-062607, and fostamatinib.

Further examples of BTK inhibitory compounds, and conditions treatableby such compounds in combination with compounds of this invention can befound in WO 2008/039218 and WO 2011/090760, the entirety of which areincorporated herein by reference.

Further examples of SYK inhibitory compounds, and conditions treatableby such compounds in combination with compounds of this invention can befound in WO 2003/063794, WO 2005/007623, and WO 2006/078846, theentirety of which are incorporated herein by reference.

Further examples of PI3K inhibitory compounds, and conditions treatableby such compounds in combination with compounds of this invention can befound in WO 2004/019973, WO 2004/089925, WO 2007/016176, U.S. Pat. No.8,138,347, WO 2002/088112, WO 2007/084786, WO 2007/129161, WO2006/122806, WO 2005/113554, and WO 2007/044729 the entirety of whichare incorporated herein by reference.

Further examples of JAK inhibitory compounds, and conditions treatableby such compounds in combination with compounds of this invention can befound in WO 2009/114512, WO 2008/109943, WO 2007/053452, WO 2000/142246,and WO 2007/070514, the entirety of which are incorporated herein byreference.

Further anti-angiogenic compounds include compounds having anothermechanism for their activity, e.g., unrelated to protein or lipid kinaseinhibition, e.g., thalidomide (Thalomid™) and TNP-470.

Examples of proteasome inhibitors useful for use in combination withcompounds of the invention include, but are not limited to bortezomib,disulfiram, epigallocatechin-3-gallate (EGCG), salinosporamide A,carfilzomib, ONX-0912, CEP-18770, and MLN9708.

Compounds which target, decrease or inhibit the activity of a protein orlipid phosphatase are, e.g., inhibitors of phosphatase 1, phosphatase2A, or CDCl₂5, such as okadaic acid or a derivative thereof.

Compounds which induce cell differentiation processes include, but arenot limited to, retinoic acid, α- γ- or δ-tocopherol or α- γ- orδ-tocotrienol.

The term cyclooxygenase inhibitor as used herein includes, but is notlimited to, Cox-2 inhibitors, 5-alkyl substituted2-arylaminophenylacetic acid and derivatives, such as celecoxib(Celebrex™), rofecoxib (Vioxx™), etoricoxib, valdecoxib or a5-alkyl-2-arylaminophenylacetic acid, such as5-methyl-2-(2′-chloro-6′-fluoroanilino)phenyl acetic acid, lumiracoxib.

The term “bisphosphonates” as used herein includes, but is not limitedto, etridonic, clodronic, tiludronic, pamidronic, alendronic,ibandronic, risedronic and zoledronic acid. Etridonic acid is marketedunder the trade name Didronel™. Clodronic acid is marketed under thetrade name Bonefos™. Tiludronic acid is marketed under the trade nameSkelid™. Pamidronic acid is marketed under the trade name Aredia™.Alendronic acid is marketed under the trade name Fosamax™. Ibandronicacid is marketed under the trade name Bondranat™. Risedronic acid ismarketed under the trade name Actonel™. Zoledronic acid is marketedunder the trade name Zometa™. The term “mTOR inhibitors” relates tocompounds which inhibit the mammalian target of rapamycin (mTOR) andwhich possess antiproliferative activity such as sirolimus (Rapamune®),everolimus (Certican™), CCI-779 and ABT578.

The term “heparanase inhibitor” as used herein refers to compounds whichtarget, decrease or inhibit heparin sulfate degradation. The termincludes, but is not limited to, PI-88. The term “biological responsemodifier” as used herein refers to a lymphokine or interferons.

The term “inhibitor of Ras oncogenic isoforms”, such as H-Ras, K-Ras, orN-Ras, as used herein refers to compounds which target, decrease orinhibit the oncogenic activity of Ras; for example, a “farnesyltransferase inhibitor” such as L-744832, DK8G557 or R115777 (Zamestra™).The term “telomerase inhibitor” as used herein refers to compounds whichtarget, decrease or inhibit the activity of telomerase. Compounds whichtarget, decrease or inhibit the activity of telomerase are especiallycompounds which inhibit the telomerase receptor, such as telomestatin.

The term “methionine aminopeptidase inhibitor” as used herein refers tocompounds which target, decrease or inhibit the activity of methionineaminopeptidase. Compounds which target, decrease or inhibit the activityof methionine aminopeptidase include, but are not limited to, bengamideor a derivative thereof.

The term “proteasome inhibitor” as used herein refers to compounds whichtarget, decrease or inhibit the activity of the proteasome. Compoundswhich target, decrease or inhibit the activity of the proteasomeinclude, but are not limited to, Bortezomib (Velcade™) and MLN 341.

The term “matrix metalloproteinase inhibitor” or (“MMP” inhibitor) asused herein includes, but is not limited to, collagen peptidomimetic andnonpeptidomimetic inhibitors, tetracycline derivatives, e.g.,hydroxamate peptidomimetic inhibitor batimastat and its orallybioavailable analogue marimastat (BB-2516), prinomastat (AG3340),metastat (NSC 683551) BMS-279251, BAY 12-9566, TAA211, MMI270B orAAJ996.

The term “compounds used in the treatment of hematologic malignancies”as used herein includes, but is not limited to, FMS-like tyrosine kinaseinhibitors, which are compounds targeting, decreasing or inhibiting theactivity of FMS-like tyrosine kinase receptors (Flt-3R); interferon,1-O-D-arabinofuransylcytosine (ara-c) and bisulfan; and ALK inhibitors,which are compounds which target, decrease or inhibit anaplasticlymphoma kinase.

Compounds which target, decrease or inhibit the activity of FMS-liketyrosine kinase receptors (Flt-3R) are especially compounds, proteins orantibodies which inhibit members of the Flt-3R receptor kinase family,such as PKC412, midostaurin, a staurosporine derivative, SU11248 andMLN518.

The term “HSP90 inhibitors” as used herein includes, but is not limitedto, compounds targeting, decreasing or inhibiting the intrinsic ATPaseactivity of HSP90; degrading, targeting, decreasing or inhibiting theHSP90 client proteins via the ubiquitin proteosome pathway. Compoundstargeting, decreasing or inhibiting the intrinsic ATPase activity ofHSP90 are especially compounds, proteins or antibodies which inhibit theATPase activity of HSP90, such as 17-allylamino,17-demethoxygeldanamycin(17AAG), a geldanamycin derivative; other geldanamycin relatedcompounds; radicicol and HDAC inhibitors.

The term “antiproliferative antibodies” as used herein includes, but isnot limited to, trastuzumab (Herceptin™), Trastuzumab-DM1, erbitux,bevacizumab (Avastin™) rituximab (Rituxan®), PRO64553 (anti-CD40) and2C4 Antibody. By antibodies is meant intact monoclonal antibodies,polyclonal antibodies, multispecific antibodies formed from at least 2intact antibodies, and antibodies fragments so long as they exhibit thedesired biological activity.

For the treatment of acute myeloid leukemia (AML), compounds of thecurrent invention can be used in combination with standard leukemiatherapies, especially in combination with therapies used for thetreatment of AML. In particular, compounds of the current invention canbe administered in combination with, for example, farnesyl transferaseinhibitors and/or other drugs useful for the treatment of AML, such asDaunorubicin, Adriamycin, Ara-C, VP-16, Teniposide, Mitoxantrone,Idarubicin, Carboplatinum and PKC412.

Other anti-leukemic compounds include, for example, Ara-C, a pyrimidineanalog, which is the 2′-alpha-hydroxy ribose (arabinoside) derivative ofdeoxycytidine. Also included is the purine analog of hypoxanthine,6-mercaptopurine (6-MP) and fludarabine phosphate. Compounds whichtarget, decrease or inhibit activity of histone deacetylase (HDAC)inhibitors such as sodium butyrate and suberoylanilide hydroxamic acid(SAHA) inhibit the activity of the enzymes known as histonedeacetylases. Specific HDAC inhibitors include MS275, SAHA, FK228(formerly FR901228), Trichostatin A and compounds disclosed in U.S. Pat.No. 6,552,065 including, but not limited to,N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide, or a pharmaceutically acceptablesalt thereof andN-hydroxy-3-[4-[(2-hydroxyethyl){2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide, or a pharmaceutically acceptable salt thereof, especiallythe lactate salt. Somatostatin receptor antagonists as used herein referto compounds which target, treat or inhibit the somatostatin receptorsuch as octreotide, and SOM230. Tumor cell damaging approaches refer toapproaches such as ionizing radiation. The term “ionizing radiation”referred to above and hereinafter means ionizing radiation that occursas either electromagnetic rays (such as X-rays and gamma rays) orparticles (such as alpha and beta particles). Ionizing radiation isprovided in, but not limited to, radiation therapy and is known in theart (see Hellman, Principles of Radiation Therapy, Cancer, in Principlesand Practice of Oncology, Devita et al., Eds., 4^(th) Edition, Vol. 1,pp. 248-275 (1993)).

Also included are EDG binders and ribonucleotide reductase inhibitors.The term “EDG binders” as used herein refers to a class ofimmunosuppressants that modulates lymphocyte recirculation, such asFTY720. The term “ribonucleotide reductase inhibitors” refers topyrimidine or purine nucleoside analogs including, but not limited to,fludarabine and/or cytosine arabinoside (ara-C), 6-thioguanine,5-fluorouracil, cladribine, 6-mercaptopurine (especially in combinationwith ara-C against ALL) and/or pentostatin. Ribonucleotide reductaseinhibitors are especially hydroxyurea or2-hydroxy-1H-isoindole-1,3-dione derivatives.

Also included are in particular those compounds, proteins or monoclonalantibodies of VEGF such as1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine or a pharmaceuticallyacceptable salt thereof,1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine succinate;Angiostatin™; Endostatin™; anthranilic acid amides; ZD4190; ZD6474;SU5416; SU6668; bevacizumab; or anti-VEGF antibodies or anti-VEGFreceptor antibodies, such as rhuMAb and RHUFab, VEGF aptamer such asMacugon; FLT-4 inhibitors, FLT-3 inhibitors, VEGFR-2 IgGI antibody,Angiozyme (RPI 4610) and Bevacizumab (Avastin™)

Photodynamic therapy as used herein refers to therapy which uses certainchemicals known as photosensitizing compounds to treat or preventcancers. Examples of photodynamic therapy include treatment withcompounds, such as Visudyne™ and porfimer sodium.

Angiostatic steroids as used herein refers to compounds which block orinhibit angiogenesis, such as, e.g., anecortave, triamcinolone,hydrocortisone, 11-α-epihydrocortisol, cortexolone,17α-hydroxyprogesterone, corticosterone, desoxycorticosterone,testosterone, estrone and dexamethasone.

Implants containing corticosteroids refers to compounds, such asfluocinolone and dexamethasone.

Other chemotherapeutic compounds include, but are not limited to, plantalkaloids, hormonal compounds and antagonists; biological responsemodifiers, preferably lymphokines or interferons; antisenseoligonucleotides or oligonucleotide derivatives; shRNA or siRNA; ormiscellaneous compounds or compounds with other or unknown mechanism ofaction.

The structure of the active compounds identified by code numbers,generic or trade names may be taken from the actual edition of thestandard compendium “The Merck Index” or from databases, e.g., PatentsInternational (e.g., IMS World Publications).

A compound of the current invention may also be used in combination withknown therapeutic processes, for example, the administration of hormonesor radiation. In certain embodiments, a provided compound is used as aradiosensitizer, especially for the treatment of tumors which exhibitpoor sensitivity to radiotherapy.

A compound of the current invention can be administered alone or incombination with one or more other therapeutic compounds, possiblecombination therapy taking the form of fixed combinations or theadministration of a compound of the invention and one or more othertherapeutic compounds being staggered or given independently of oneanother, or the combined administration of fixed combinations and one ormore other therapeutic compounds. A compound of the current inventioncan besides or in addition be administered especially for tumor therapyin combination with chemotherapy, radiotherapy, immunotherapy,phototherapy, surgical intervention, or a combination of these.Long-term therapy is equally possible as is adjuvant therapy in thecontext of other treatment strategies, as described above. Otherpossible treatments are therapy to maintain the patient's status aftertumor regression, or even chemopreventive therapy, for example inpatients at risk.

Those additional agents may be administered separately from an inventivecompound-containing composition, as part of a multiple dosage regimen.Alternatively, those agents may be part of a single dosage form, mixedtogether with a compound of this invention in a single composition. Ifadministered as part of a multiple dosage regime, the two active agentsmay be submitted simultaneously, sequentially or within a period of timefrom one another normally within five hours from one another.

As used herein, the term “combination,” “combined,” and related termsrefers to the simultaneous or sequential administration of therapeuticagents in accordance with this invention. For example, a compound of thepresent invention may be administered with another therapeutic agentsimultaneously or sequentially in separate unit dosage forms or togetherin a single unit dosage form. Accordingly, the present inventionprovides a single unit dosage form comprising a compound of the currentinvention, an additional therapeutic agent, and a pharmaceuticallyacceptable carrier, adjuvant, or vehicle.

The amount of both an inventive compound and additional therapeuticagent (in those compositions which comprise an additional therapeuticagent as described above) that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. Preferably,compositions of this invention should be formulated so that a dosage ofbetween 0.01-100 mg/kg body weight/day of an inventive compound can beadministered.

In those compositions which comprise an additional therapeutic agent,that additional therapeutic agent and the compound of this invention mayact synergistically. Therefore, the amount of additional therapeuticagent in such compositions will be less than that required in amonotherapy utilizing only that therapeutic agent. In such compositionsa dosage of between 0.01-1,000 μg/kg body weight/day of the additionaltherapeutic agent can be administered.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

In some embodiments, the additional therapeutic agent administered incombination with a compound of the present invention is another mTORinhibitor. In some embodiments, the additional mTOR inhibitor inhibitsmTOR by binding the catalytic active site of mTOR. Examples of suchadditional mTOR inhibitors include: dactolisib,8-(6-methoxy-pyridin-3-yl)-3-methyl-1-(4-piperazin-1-yl-3-trifluoromethyl-phenyl)-1,3-dihydro-imidazo[4,5-c]quinolin-2-one(WO 2006/122806), vistusertib (AZD2014; WO 2009/153597); AZD8055 (WO2009/153597; XL388 (U.S. Pat. App. Pub. 2010/0305093); sapanisertib(MLN0128; INK128; WO 2015/051043); DS3078; apitolisib (GDC0980; WO2008/070740); omipalisib (GSK-2126458; WO 2008/14446); NVP-BGT226(Chang, K. Y., et al., Clin. Cancer Res. 17(22): 7116-26 (2011));voxtalisib (XL765; SAR245409; WO 2007/044813); PF04691502 (WO2008/032162); gedatolisib (PF05212384; PKI-587; WO 2009/143313); SF1126(WO 2004/089925); GSK1059615 (WO 2007/136940); BI-860585; OSI 027 (WO2007/061737); VS 5584 (WO 2010/114484); CC-223 (WO 2010/062571);DCBCI-0901 (Lee, Y. E., et al., Mol. Canc. Thera. 12(11 Suppl): Abstractnr C270 (2013)):); LY3023414 (WO 2012/097039); P529 (WO 2007/133249);panulisib (P7170; WO 2012/007926); DS-7423 (Kashiyama, T., et al., PLoSOne 9(2): e87220 (2014)); PWT33567 mesylate (VCD-597; WO 2010/110685);ME-344 (NV-128; Navarro, P., et al., Cell Rep. 15(12):2705-18 (2016));ABTLO812 (WO 2010/106211); WYE-132; EXEL-3885 (Eur J Cancer Suppl.6(12): Abst 322 (2008)); EXEL-4431 (Eur J Cancer Suppl. 6(12): Abst 322(2008)); AR-mTOR-26 (101st Annu Meet Am Assoc Cancer Res (AACR) (April17-21, Washington, D.C.) 2010, Abst 4484); NV-128 (A. B. Alvero et al.,Mol Cancer Ther. 10(8): 1385-93 (2011)); salinomycin (VS-507; Gupta, P.B., et al., Cell 138(4): 645-59 (2009)); BN-107; BN-108; WAY-600;WYE-687; WYE-354 (Yu, K., et al., Cancer Res. 69(15): 6232-40 (2009));Ku-063794 (Garcia-Martinez, J. M., et al., Biochem. J. 421(1): 29-42(2009)); torkinib (PP242; Apsel, B., et al., Nat. Chem. Biol. 4(11):691-99 (2008)); PP30; CZ415 (REF); INK1069; EXEL-2044; EXEL-7518;SB2158; SB2280; AR-mTOR-1 (Wallace, E. M., et al., Mol. Canc. Thera.8(12 Suppl): Abst. B267 (2009)).

Reference to any particular additional mTOR inhibitor herein alsocomprises any pharmaceutically acceptable salts, stereoisomers,tautomers, solvates, hydrates and polymorphs thereof.

The compounds of this invention, or pharmaceutical compositions thereof,may also be incorporated into compositions for coating an implantablemedical device, such as prostheses, artificial valves, vascular grafts,stents and catheters. Vascular stents, for example, have been used toovercome restenosis (re-narrowing of the vessel wall after injury).However, patients using stents or other implantable devices risk clotformation or platelet activation. These unwanted effects may beprevented or mitigated by pre-coating the device with a pharmaceuticallyacceptable composition comprising a kinase inhibitor. Implantabledevices coated with a compound of this invention are another embodimentof the present invention.

EXEMPLIFICATION

As depicted in the Examples below, in certain exemplary embodiments,compounds are prepared according to the following general procedures. Itwill be appreciated that, although the general methods depict thesynthesis of certain compounds of the present invention, the followinggeneral methods, and other methods known to one of ordinary skill in theart, can be applied to all compounds and subclasses and species of eachof these compounds, as described herein (see also Luengo, J. I. et al.,Chem. Biol., 2(7): 471-81 (1995); and Grinfeld, A. A. et al., Tet.Lett., 35(37): 6835-38 (1994)).

List of Abbrevations Used in the Experimental Section

CH₃CN: acetonitrile

DCE: dichloroethane

DCM: dichloromethane

DIPEA: N,N-diisopropylethylamine

DMF: N,N-dimethylformamide

DMSO: dimethyl sulfoxide

ESI: electrospray ionization

EtOAc: ethyl acetate

EtOH: ethanol

h: hours

HBr: hydrogen bromide

HF: hydrogen fluoride

HND-8: acidic ion exchange resin (e.g., Amberlyst)

H2O: water

HPLC: high performance liquid chromatography

MeOH: methanol

min: minutes

mL: milliliters

mM: millimolar

mmol: millimoles

MS: mass spectrometry

N₂: nitrogen gas

NaHCO₃: sodium bicarbonate

NaI: sodium iodide

NaN₃. sodium azide

NaOH: sodium hydroxide

Na₂SO₄: sodium sulfate

NH₄Cl: ammonium chloride

NMR: Nuclear Magnetic Resonance

° C.: degrees Celsius

prep-HPLC: preparative high performance liquid chromatography

PPh₃: triphenylphosphine

p-TsOH: para toluenesulfonic acid

rt: room temperature

TEA: triethylamine

TFA: trifluoracetic acid

THF: tetrahydrofuran

Example 1: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-28, I-29 and I-30)

Synthetic Scheme:

Procedures and Characterization:

2-[2-(2-hydroxyethoxy) ethoxy] ethanol (5 mL) was added to a solution ofrapamycin (0.5 g, 0.547 mmol) and p-toluenesulfonic acid hydrate (0.52g, 2.73 mmol) in THF (15 mL) at 25° C. The resulting mixture was stirredfor 2 hours, then added to an ice cold saturated NaHCO₃ aqueous solutionand extracted with EtOAc (30 mL×3). The organic layers were combinedthen dried over Na₂SO₄, filtered, and concentrated in vacuo. The crudematerial was purified by reverse phase chromatography (CH₃CN/pure water:7:3) to obtain(21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-28: 0.19 g, 33.7% yield) as a white solid. MS (EI⁺, m/z): 1054.4[M+Na]⁺. 1.5 g of this material was sent for chiral separation whichprovided I-29 (0.6 g) and I-30 (0.2 g).

The chiral separation method:

Column: CHIRALPAK IC (IC00CD-TB016)

Column size: 0.46 cm I.D.×15 cm L

Mobile phase: Hexane/EtOH=60/40 (V/V)

Flow rate: 1.0 ml/min

Wave length: UV 254 nm

Temperature: 35° C.

HPLC equipment: Shimadzu LC-20AD CP-HPLC-05

I-29: ¹H NMR (500 MHz, CDCl₃) δ 6.41-6.20 (m, 2H), 6.13 (dd, J=15.0,10.3 Hz, 1H), 5.92 (dd, J=32.7, 11.0 Hz, 1H), 5.51 (dd, J=15.1, 8.9 Hz,1H), 5.41 (d, J=9.9 Hz, 1H), 5.27 (d, J=5.3 Hz, 1H), 5.13 (dd, J=26.5,20.5 Hz, 1H), 4.85 (s, 1H), 4.19 (t, J=8.9 Hz, 1H), 3.92 (d, J=36.4 Hz,1H), 3.80-3.51 (m, 12H), 3.50-3.24 (m, 12H), 2.87-2.51 (m, 6H), 2.29 (t,J=34.7 Hz, 2H), 2.12-1.87 (m, 5H), 1.84-1.66 (m, 13H), 1.53-1.15 (m,9H), 1.15-0.77 (m, 18H), 0.65 (dt, J=20.2, 10.1 Hz, 1H).

Example 2: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-43-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]-45,55-dihydroxy-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentonetrifluoroacetic acid salt (I-39)

Synthetic Scheme:

Procedures and Characterization: Step 1:2-(2-(2-bromoethoxy)ethoxy)ethanol

Hydrogen bromide (86.21 g, 1.07 mmol, 115 mL) was added to a solution of2-[2-(2-hydroxyethoxy)ethoxy]ethanol (100 g, 665.90 mmol) in toluene(1.15 L) and the resulting mixture was stirred at reflux for 18 hours,then the water layer was discarded. The organic layer was washed withaqueous NaOH solution, concentrated in vacuo, then purified by silicagel chromatography (MeOH:DCM=1:20) to obtain2-[2-(2-bromoethoxy)ethoxy]ethanol (20 g, 14% yield) as a liquid. ¹H NMR(400 MHz, CDCl₃) δ 3.83 (t, J=6.2 Hz, 2H), 3.77-3.72 (m, 2H), 3.69 (s,4H), 3.64-3.61 (m, 2H), 3.49 (t, J=6.1 Hz, 2H), 2.51 (t, J=6.1 Hz, 1H).

Step 2:(21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-43-[2-[2-(2-bromoethoxy)ethoxy]ethoxy]-45,55-dihydroxy-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone

2-[2-(2-bromoethoxy)ethoxy]ethanol (0.12 g, 0.547 mmol, 2 mL) was addedto a solution of rapamycin (0.5 g, 0.547 mmol) and p-toluenesulfonicacid hydrate (0.5 g, 2.73 mmol) in THF (7 mL) at room temperature andthe resulting mixture was stirred for 2 hours. Ice cold NaHCO₃ aqueoussolution was then added and the mixture was extracted with EtOAc (30mL×3). The organic phase was then dried over Na₂SO₄, filtered, andconcentrated in vacuo. The resulting crude material was purified byreverse phase chromatography (CH₃CN/pure water=7:3) to obtain(21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-43-[2-[2-(2-bromoethoxy)ethoxy]ethoxy]-45,55-dihydroxy-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(0.2 g, 33.4% yield, ¹HNMR shows a rapamycin impurity) as a white solid.MS (EI⁺, m/z): 1116.4 [M+Na]⁺.

Step 3:(21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-43-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]-45,55-dihydroxy-44-[(S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-68,69-dioxa-59-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone

A solution of NaN₃ (1.07 g, 16.44 mmol), NaI (0.33 g, 2.19 mmol) and(21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-43-[2-[2-(2-bromoethoxy)ethoxy]ethoxy]-45,55-dihydroxy-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(0.6 g, 0.548 mmol) in DMF (10 mL) was stirred at 60° C. for 1.5 hours.The reaction was then quenched by EtOAc (50 mL) and the mixture washedwith NH₄Cl aqueous solution (20 mL), dried over Na₂SO₄, filtered andconcentrated in vacuo. The resulting crude material was purified byreverse phase chromatography (CH₃CN/pure water=4:1) to obtain(21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-43-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]-45,55-dihydroxy-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-68,69-dioxa-59-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(0.3g, 51.8% yield) as a light yellow solid. MS (EI⁺, m/z): 1079.4[M+Na]⁺.

Step 4:(21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-43-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]-45,55-dihydroxy-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentoneTrifluoroacetic Acid Salt

Triphenylphoshine (0.186 g, 0.7 mmol) was added slowly to a solution of(21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-43-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]-45,55-dihydroxy-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-68,69-dioxa-59-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(0.25 g, 0.24 mmol) in THF (5 mL) triphenylphosphine. The resultingsolution was stirred at 60° C. for 2 hours, and then 0.05 mL of waterwas added and the mixture stirred at room temperature for 6 hours, thenconcentrated. The resulting crude material was purified by reverse phasechromatography (CH₃CN/0.02% TFA in water (2:3) to obtain(21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-43-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]-45,55-dihydroxy-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-39: 0.035 g, 14% yield) as a white solid. MS (EI⁺, m/z):1031.4[M+H]⁺. ¹H NMR (500 MHz, DMSO-d₆) δ 7.82 (s, 3H), 6.42 (dd,J=33.7, 19.3 Hz, 2H), 6.25-6.09 (m, 2H), 5.46 (dd, J=14.7, 9.7 Hz, 1H),5.28 (s, 1H), 5.08 (d, J=10.1 Hz, 1H), 5.00-4.92 (m, 1H), 4.08-3.92 (m,2H), 3.78 (d, J=11.6 Hz, 1H), 3.63-3.38 (m, 16H), 3.36-3.08 (m, 12H),2.99 (dd, J=22.3, 17.1 Hz, 2H), 2.87-2.73 (m, 2H), 2.37 (dd, J=17.9, 8.4Hz, 1H), 2.30-1.75 (m, 4H), 1.7-1.49 (m, 15H), 1.51-1.01 (m, 6H),1.01-0.65 (m, 18H), 0.63-0.56 (m, 1H).

Example 3: Synthesis of(21E,23E,25E,26E,36R,37S,38R,39R,41R,43S,46S,47R,48R,57S)-47,57-dihydroxy-45-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-46-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone(I-36)

Synthetic Scheme:

Procedures and Characterization:

2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethanol (1.06 g, 5.47 mmol, 5 mL)was added to a solution of rapamycin (0.5 g, 0.547 mmol) andp-toluenesulfonic acid hydrate (0.21 g, 1.09 mmol) in THF (15 mL) atroom temperature. The reaction mixture was stirred at room temperaturefor 2 hours, then added to ice cold saturated NaHCO₃ aqueous solutionand extracted with EtOAc (30 mL×3). The organic layers were the combinedand dried over Na₂SO₄, filtered and concentrated. The crude was purifiedby reverse phase chromatography (CH₃CN/pure water=3:2) to obtain(21E,23E,25E,26E,36R,37S,38R,39R,41R,43S,46S,47R,48R,57S)-47,57-dihydroxy-45-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-46-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone(I-36: 0.15 g, 25.5% yield) as a white solid. MS (EI⁺, m/z):1098.4[M+H]⁺. ¹H NMR (500 MHz, CDCl₃) δ 6.40-5.92 (m, 4H), 5.73-5.35 (m,3H), 5.25-5.05 (m, 2H), 4.31-4.12 (m, 1H), 3.97 (dd, J=25.7, 6.3 Hz,1H), 3.87-3.53 (m, 15H), 3.52-3.17 (m, 11H), 2.99-2.46 (m, 6H),2.36-1.93 (m, 9H), 1.90-1.54 (m, 13H), 1.52-1.17 (m, 9H), 1.15-0.81 (m,18H), 0.68-0.58 (m, 1H).

Example 4: Synthesis of(21E,23E,25E,26E,38R,39S,40R,41R,43R,45S,47R,48S,49R,50R,59S)-49,59-dihydroxy-47-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]-48-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-50-methoxy-38,39,40,41,51,52-hexamethyl-70,71-dioxa-60-azatricyclohexatriaconta-21,23,25(51),26(52)-tetraene-53,54,55,56,57-pentone(I-35)

Synthetic Scheme:

Procedures and Characterization:

2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethanol (0.08 g, 0.33mmol, 2 mL) was added to a solution of rapamycin (0.3 g, 0.328 mmol) andp-toluenesulfonic acid hydrate (0.31 g, 1.64 mmol) in THF (6 mL) at roomtemperature. The resulting mixture was stirred at room temperature for 2hours, then added to ice cold saturated NaHCO₃ aqueous solution andextracted with EtOAc (20 mL×3). The organic layers were combined andthen dried over Na₂SO₄, filtered, and concentrated in vacuo. The crudematerial was purified by reverse phase chromatography (CH₃CN/pure water:3:2) to obtain(21E,23E,25E,26E,38R,39S,40R,41R,43R,45S,47R,48S,49R,50R,59S)-49,59-dihydroxy-47-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]-48-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-50-methoxy-38,39,40,41,51,52-hexamethyl-70,71-dioxa-60-azatricyclohexatriaconta-21,23,25(51),26(52)-tetraene-53,54,55,56,57-pentone(I-35: 0.06 g, 16.3% yield) as a white solid. MS (EI⁺, m/z):1042.4[M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ 6.42-5.81 (m, 4H), 5.58-4.81(m, 4H), 4.31-4.11 (m, 1H), 4.01-3.51 (m, 22H), 3.49-3.13 (m, 11H),3.01-2.43 (m, 6H), 2.29 (t, J=30.6 Hz, 2H), 2.15-1.88 (m, 7H), 1.76-1.55(m, 12H), 1.51-1.18 (m, 9H), 1.15-0.74 (m, 18H), 0.66 (dd, J=23.9, 12.0Hz, 1H).

Example 5: Synthesis of(21E,23E,25E,26E,30R,31S,32R,33R,35R,37S,39S,40S,41R,42R,52S)-39-(2,3-dihydroxypropoxy)-41,52-dihydroxy-40-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-42-methoxy-30,31,32,33,43,44-hexamethyl-64,65-dioxa-53-azatricyclohexatriaconta-21,23,25(43),26(44)-tetraene-45,46,47,48,49-pentone(I-25)

Synthetic Scheme:

Procedures and Characterization: Step 1:(24E,26E,28E,29E,32R,33S,34R,35R,37R,39S,41S,42S,44R,45R,55S)-41-[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]-44,55-dihydroxy-42-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-32,33,34,35,46,47-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-24,26,28(46),29(47)-tetraene-48,49,50,51,52-pentone

(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (1 mL) was added to a solutionof rapamycin (0.2 g, 0.22 mmol) and p-toluenesulfonic acid hydrate (0.1g, 0.547 mmol) in THF (3 mL) at room temperature. The resulting mixturestirred at room temperature for 2 hours, then added to ice coldsaturated NaHCO₃ aqueous solution and extracted with EtOAc (20 mL×2).The organic layers were combined and then dried over Na₂SO₄, filtered,and concentrated in vacuo. The crude was purified by reverse phasechromatography (CH₃CN/pure water: 7:3) to obtain(24E,26E,28E,29E,32R,33S,34R,35R,37R,39S,41S,42S,44R,45R,55S)-41-[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]-44,55-dihydroxy-42-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-32,33,34,35,46,47-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-24,26,28(46),29(47)-tetraene-48,49,50,51,52-pentone(0.07 g, 32% yield) as a white solid. MS (EI⁺, m/z): 1036.4[M+Na]⁺.

Step 2:(21E,23E,25E,26E,30R,31S,32R,33R,35R,37S,39S,40S,41R,42R,52S)-39-(2,3-dihydroxypropoxy)-41,52-dihydroxy-40-[(S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-42-methoxy-30,31,32,33,43,44-hexamethyl-64,65-dioxa-53-azatricyclohexatriaconta-21,23,25(43),26(44)-tetraene-45,46,47,48,49-pentone(I-25)

4-methylbenzenesulfonic acid-pyridine (0.037 g, 0.148 mmol) was added toa solution of(24E,26E,28E,29E,32R,33S,34R,35R,37R,39S,41S,42S,44R,45R,55S)-41-[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]-44,55-dihydroxy-42-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-32,33,34,35,46,47-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-24,26,28(46),29(47)-tetraene-48,49,50,51,52-pentone(0.05 g, 0.05 mmol) in MeOH (2 mL). The mixture was stirred at roomtemperature for 18 hours. After the evaporation of methanol, the residuewas neutralized with saturated aqueous NaHCO₃. The mixture was thenextracted with EtOAc (10 mL×3). The organic layers were combined, driedover Na₂SO₄, and concentrated with the resulting crude material purifiedby reverse phase chromatography (CH₃CN/pure water=1:1) to give(21E,23E,25E,26E,30R,31S,32R,33R,35R,37S,39S,40S,41R,42R,52S)-39-(2,3-dihydroxypropoxy)-41,52-dihydroxy-40-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-42-methoxy-30,31,32,33,43,44-hexamethyl-64,65-dioxa-53-azatricyclohexatriaconta-21,23,25(43),26(44)-tetraene-45,46,47,48,49-pentone(I-25: 0.005 g, 10% yield) as a white solid. MS (EI⁺, m/z):996.5[M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ 6.21-5.97 (m, 4H), 5.77-4.78 (m,5H), 4.53-4.12 (m, 2H), 4.05-3.11 (m, 19H), 3.07-2.88 (m, 2H), 2.65-2.5(m, 4H), 2.39-1.91 (m, 7H), 1.89-1.69 (m, 12H), 1.52-1.20 (m, 9H),1.17-0.76 (m, 18H), 0.73-0.61 (m, 1H).

Example 6: Synthesis of(21E,23E,25E,26E,42R,43S,44R,45R,47S,49S,51S,52S,53R,54R,63R)-53,63-dihydroxy-51-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-52-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-54-methoxy-42,43,44,45,55,56-hexamethyl-74,75-dioxa-64-azatricyclohexatriaconta-21,23,25(55),26(56)-tetraene-57,58,59,60,61-pentone(I-24)

Synthetic Scheme:

Procedures and Characterization:

A solution of rapamycin (0.5 g, 547 mmol) and2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol(0.2 g, 0.547 mmol, 3 mL) and p-toluenesulfonic acid hydrate (0.52 g,2.73 mmol) in THF (15 mL) was stirred at room temperature for 3 hours,then quenched with EtOAc (30 mL×3). The combined organic layers werewashed with ice cold saturated NaHCO₃ solution and concentrated. Theresulting crude material was purified by reverse phase chromatography(CH₃CN/pure water=7:3) to obtain(21E,23E,25E,26E,42R,43S,44R,45R,47S,49S,51S,52S,53R,54R,63R)-53,63-dihydroxy-51-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-52-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-54-methoxy-42,43,44,45,55,56-hexamethyl-74,75-dioxa-64-azatricyclohexatriaconta-21,23,25(55),26(56)-tetraene-57,58,59,60,61-pentone(I-24: 0.13 g, 19%) as a white solid. MS (EI⁺, m/z): 1275.6[M+Na]⁺. ¹HNMR (500 MHz, CDCl₃) δ 6.45-5.80 (m, 4H), 5.57-5.05 (m, 4H), 4.85-4.08(m, 2H), 3.90-3.50 (m, 34H), 3.47-3.24 (m, 13H), 2.97-2.44 (m, 7H),2.39-2.06 (m, 2H), 2.03-1.84 (m, 6H), 1.78-1.58 (m, 13H), 1.53-1.16 (m,9H), 1.14-0.78 (m, 18H), 0.67-0.53 (m, 1H).

Example 7: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,38S,41S,43S,45R,46R,55R)-45,55-dihydroxy-42-[2-(2-hydroxyethoxy)ethoxy]-43-[(1R)-2-[(1S,2R,3R)-3-(2-hydroxyethoxy)-2-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-23)

Synthetic Scheme:

Procedures and Characterization: Step 1:2-[tert-butyl(dimethyl)silyl]oxyethyl Trifluoromethanesulfonate

A mixture of 2-[tert-butyl(dimethyl)silyl] oxyethanol (4 g, 22.69 mmol)and DIPEA (3.81 g, 29.49 mmol, 5.14 mL) in DCM (50 mL) was cooled to 0°C. under N₂, then trifluoromethylsulfonyl trifluoromethanesulfonate(7.04 g, 24.95 mmol) was added and the resulting mixture was stirred at0° C. for 1 hour. The reaction was diluted with EtOAc (200 mL) thenwashed with saturated. NaHCO₃(200 mL), water (200 mL), brine (200 mL),and then the organic layer was dried over Na₂SO₄, filtered, andconcentrated in vacuo to afford 2-[tert-butyl(dimethyl)silyl]oxyethyltrifluoromethanesulfonate (5.5 g, 78.6% yield) as a brown oil. ¹H NMR(500 MHz, CDCl₃) δ 4.50-4.42 (t, 2H), 3.85-3.80 (t, 2H), 0.84-0.78 (s,9H), 0.00 (s, 6H).

Step 2:(27E,29E,31E,32E,36R,37S,38R,39R,40S,43S,44S,45S,47R,48R,57R)-45-[(1R)-2-[(1S,2R,3R)-3-[2-[tert-butyl(dimethyl)silyl]oxyethoxy]-2-methoxy-cyclohexyl]-1-methyl-ethyl]-47,57-dihydroxy-44,48-dimethoxy-36,37,38,39,49,50-hexamethyl-67,68-dioxa-59-azatricyclohexatriaconta-27,29,31(49),32(50)-tetraene-51,52,53,54,55-pentone

Rapamycin (2 g, 2.19 mmol) and DIPEA (2.26 g, 17.50 mmol, 3.05 mL) weredissolved in toluene (60 mL) then heated to 60° C.2-[tert-butyl(dimethyl)silyl]oxyethyl trifluoromethanesulfonate (5.40 g,17.50 mmol) was then added under N₂, then stirred at 60° C. for 16hours. The mixture was poured into ice cold saturated NaHCO₃ (100 mL)and then extracted with EtOAc (150 mL×3). The combined organic layerswere washed with water and brine, then concentrated in vacuo. Theresidue was then purified by reverse phase chromatography (CH₃CN/purewater=4:1) to afford(27E,29E,31E,32E,36R,37S,38R,39R,40S,43S,44S,45S,47R,48R,57R)-45-[(1R)-2-[(1S,2R,3R)-3-[2-[tert-butyl(dimethyl)silyl]oxyethoxy]-2-methoxy-cyclohexyl]-1-methyl-ethyl]-47,57-dihydroxy-44,48-dimethoxy-36,37,38,39,49,50-hexamethyl-67,68-dioxa-59-azatricyclohexatriaconta-27,29,31(49),32(50)-tetraene-51,52,53,54,55-pentone(0.75 g, 32% yield) as a colorless oil. ESI-MS (EI⁺, m/z): 1095.5[M+Na]⁺.

Step 3:(22E,24E,26E,27E,31R,32S,33R,34R,35S,38S,39S,40S,42R,43R,52R)-42,52-dihydroxy-40-[(1R)-2-[(1S,2R,3R)-3-(2-hydroxyethoxy)-2-methoxy-cyclohexyl]-1-methyl-ethyl]-39,43-dimethoxy-31,32,33,34,44,45-hexamethyl-62,63-dioxa-53-azatricyclohexatriaconta-22,24,26(44),27(45)-tetraene-46,47,48,49,50-pentone

A solution of TEA-3HF (4.65 g, 28.87 mmol) and(27E,29E,31E,32E,36R,37S,38R,39R,40S,43S,44S,45S,47R,48R,57R)-45-[(1R)-2-[(1S,2R,3R)-3-[2-[tert-butyl(dimethyl)silyl]ethoxy]-2-methoxy-cyclohexyl]-1-methyl-ethyl]-47,57-dihydroxy-44,48-dimethoxy-36,37,38,39,49,50-hexamethyl-67,68-dioxa-59-azatricyclohexatriaconta-27,29,31(49),32(50)-tetraene-51,52,53,54,55-pentone(3.05 g, 2.89 mmol) in THF (50 mL) was stirred at 20° C. for 2 hours.The mixture was poured into ice cold saturated NaHCO₃(100 mL) thenextracted with EtOAc (150 mL×3). The organic layers were combined andwashed with water and brine, then concentrated in vacuo. The residue waspurified by reverse phase chromatography (CH₃CN/pure water: 7:3) toafford(22E,24E,26E,27E,31R,32S,33R,34R,35S,38S,39S,40S,42R,43R,52R)-42,52-dihydroxy-40-[(1R)-2-[(1S,2R,3R)-3-(2-hydroxyethoxy)-2-methoxy-cyclohexyl]-1-methyl-ethyl]-39,43-dimethoxy-31,32,33,34,44,45-hexamethyl-62,63-dioxa-53-azatricyclohexatriaconta-22,24,26(44),27(45)-tetraene-46,47,48,49,50-pentone(1.5 g, 54% yield) as a white solid. ESI-MS (EI⁺, m/z): 980.5 [M+Na]⁺.

Step 4:(21E,23E,25E,26E,34R,35S,36R,37R,38S,41S,43S,45R,46R,55R)-45,55-dihydroxy-42-[2-(2-hydroxyethoxy)ethoxy]-43-[(1R)-2-[(1S,2R,3R)-3-(2-hydroxyethoxy)-2-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-23)

A mixture of(22E,24E,26E,27E,31R,32S,33R,34R,35S,38S,39S,40S,42R,43R,52R)-42,52-dihydroxy-40-[(1R)-2-[(1S,2R,3R)-3-(2-hydroxyethoxy)-2-methoxy-cyclohexyl]-1-methyl-ethyl]-39,43-dimethoxy-31,32,33,34,44,45-hexamethyl-62,63-dioxa-53-azatricyclohexatriaconta-22,24,26(44),27(45)-tetraene-46,47,48,49,50-pentone(0.5 g, 0.52 mmol), 2-(2-hydroxyethoxy)ethanol (2.77 g, 26.09 mmol) andp-toluenesulfonic acid hydrate (0.54 g, 3.13 mmol) in THF (6 mL) wasstirred at 20° C. for 2 hours. The mixture was poured into ice coldsaturated NaHCO₃ (30 mL) and extracted with EtOAc (50 mL×3). The organiclayers were combined and then washed with water and brine, thenconcentrated in vacuo. The residue was purified by reverse phasechromatography (CH₃CN/pure water=1:1) to afford(21E,23E,25E,26E,34R,35S,36R,37R,38S,41S,43S,45R,46R,55R)-45,55-dihydroxy-42-[2-(2-hydroxyethoxy)ethoxy]-43-[(1R)-2-[(1S,2R,3R)-3-(2-hydroxyethoxy)-2-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-23: 0.1 g, 19% yield) as a white solid. ESI-MS (EI⁺, m/z): 1054.4[M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ 6.63-5.86 (m, 4H), 5.70-5.00 (m, 4H),4.87-4.15 (m, 2H), 4.02-3.54 (m, 14H), 3.50-3.26 (m, 11H), 3.24-3.02 (m,3H), 2.76-2.46 (m, 3H), 2.38-1.86 (m, 8H), 1.84-1.54 (m, 14H), 1.47 (m,3H), 1.25 (m, 6H), 1.00 (m, 16H), 0.69 (dt, J=34.1, 12.1 Hz, 1H).

Example 8: Synthesis of(21E,23E,25E,26E,36R,37S,38R,39R,40S,43S,45S,47R,48R,57R)-47,57-dihydroxy-44-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-45-[(1R)-2-[(1S,2R,3R)-3-(2-hydroxyethoxy)-2-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone(I-32)

Synthetic Scheme:

Procedures and Characterization:

A mixture of everolimus (3.92 g, 26.09 mmol) and p-toluenesulfonic acidhydrate (0.45 g, 2.61 mmol) in THF (10 mL) was stirred at 20° C. for 2hours. The mixture was poured into ice cold saturated NaHCO₃ (30 mL) andextracted with EtOAc (50 mL×3). The organic layers were combined andthen washed with water and brine, then concentrated in vacuo. Theresidue was purified by reversed phase chromatography (CH₃CN/purewater=1:1) to afford(21E,23E,25E,26E,36R,37S,38R,39R,40S,43S,45S,47R,48R,57R)-47,57-dihydroxy-44-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-45-[(1R)-2-[(1S,2R,3R)-3-(2-hydroxyethoxy)-2-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone(I-32: 0.155 g, 28% yield) as a white solid. ESI-MS (EI⁺, m/z): 1098.4[M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ 6.49-5.83 (m, 4H), 5.67-5.35 (m, 2H),5.33-5.01 (m, 2H), 4.92-4.08 (m, 2H), 4.05-3.51 (m, 17H), 3.52-3.23 (m,11H), 3.23-3.01 (m, 3H), 2.66 (m, 4H), 2.40-1.95 (m, 5H), 1.95-1.55 (m,17H), 1.52-1.13 (m, 9H), 1.13-0.79 (m, 16H), 0.71 (dd, J=23.8, 11.9 Hz,1H).

Example 9: Synthesis of(21E,23E,25E,26E,38R,39S,40R,41R,42S,45S,47S,49R,50R,59R)-49,59-dihydroxy-46-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-47-[(1R)-2-[(1S,2R,3R)-3-(2-hydroxyethoxy)-2-methoxy-cyclohexyl]-1-methyl-ethyl]-50-methoxy-38,39,40,41,51,52-hexamethyl-70,71-dioxa-60-azatricyclohexatriaconta-21,23,25(51),26(52)-tetraene-53,54,55,56,57-pentone(I-22)

Synthetic Scheme:

Procedures and Characterization:

A mixture of everolimus (0.5 g, 0.52 mmol),2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethanol (2.03 g, 10.44 mmol) andp-toluenesulfonic acid hydrate (0.54 g, 3.13 mmol) in THF (6 mL) wasstirred at 20° C. for 2 h. The mixture was poured into ice coldsaturated NaHCO₃ (30 mL) and extracted with EtOAc (50 mL*3). Thecombined organic layers were washed with water, brine, then concentratedin vacuo. The residue was purified by reversed phase chromatography(CH₃CN/pure water=1:1) to afford(21E,23E,25E,26E,38R,39S,40R,41R,42S,45S,47S,49R,50R,59R)-49,59-dihydroxy-46-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-47-[(1R)-2-[(1S,2R,3R)-3-(2-hydroxyethoxy)-2-methoxy-cyclohexyl]-1-methyl-ethyl]-50-methoxy-38,39,40,41,51,52-hexamethyl-70,71-dioxa-60-azatricyclohexatriaconta-21,23,25(51),26(52)-tetraene-53,54,55,56,57-pentone(I-22: 0.1 g, 17% yield) as a white solid. ESI-MS (EI⁺, m/z): 1043.4[M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ 6.71-5.78 (m, 4H), 5.77-5.01 (m, 4H),4.65-3.88 (m, 3H), 3.87-3.50 (m, 20H), 3.50-3.00 (m, 14H), 2.77-1.95 (m,11H), 1.89-1.53 (m, 13H), 1.53-0.79 (m, 27H), 0.76-0.62 (m, 1H).

Example 10: Synthesis of(22E,24E,26E,27E,35R,36S,37R,38R,39S,42S,44S,46R,47R,56R)-46,56-dihydroxy-44-[(1R)-2-[(1S,2R,3R)-3-hydroxy-2-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-43-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]-35,36,37,38,48,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone(I-27)

Synthetic Scheme:

Procedures and Characterization:

A mixture of rapamycin (1 g, 1.09 mmol),2-[2-(2-methoxyethoxy)ethoxy]ethanol (8.98 g, 54.69 mmol) andp-toluenesulfonic acid hydrate (0.94 g, 5.47 mmol) in THF (20 mL) wasstirred at 20° C. for 2 hours. The mixture was poured into ice coldsaturated NaHCO₃ (50 mL) and extracted with EtOAc (100 mL×3). Thecombined organic layers were washed with water, brine, then concentratedin vacuo. The residue was purified by reversed phase chromatography(CH₃CN/pure water=4:1) to afford(22E,24E,26E,27E,35R,36S,37R,38R,39S,42S,44S,46R,47R,56R)-46,56-dihydroxy-44-[(1R)-2-[(1S,2R,3R)-3-hydroxy-2-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-43-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]-35,36,37,38,48,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone(I-27: 0.16 g, 14% yield) as a white solid. ESI-MS (EI⁺, m/z): 1068.4[M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ 6.50-5.81 (m, 4H), 5.73-5.05 (m, 4H),4.85-3.98 (m, 3H), 3.90-3.10 (m, 27H), 3.02-2.24 (m, 7H), 1.98 (m, 6H),1.82-1.55 (m, 13H), 1.54-1.16 (m, 9H), 1.16-0.78 (m, 17H), 0.75-0.59 (m,1H).

Example 11: Synthesis of(21E,23E,25E,26E,31R,32S,33R,34R,35S,38S,40S,42R,43R,52R)-42,52-dihydroxy-40-[(1R)-2-[(1S,2R,3R)-3-hydroxy-2-methoxy-cyclohexyl]-1-methyl-ethyl]-39-(3-hydroxypropoxy)-43-methoxy-31,32,33,34,44,45-hexamethyl-63,64-dioxa-53-azatricyclohexatriaconta-21,23,25(44),26(45)-tetraene-46,47,48,49,50-pentone(I-34)

Synthetic Scheme:

Procedures and Characterization:

A mixture of rapamycin (0.5 g, 0.55 mmol), propane-1,3-diol (13.13 g,172.48 mmol, 12.5 mL), and p-toluenesulfonic acid hydrate (0.47 g, 2.74mmol) in THF (37.5 mL) was stirred at 20° C. for 2 hours. The mixturewas poured into ice cold saturated NaHCO₃ (100 mL) and extracted withEtOAc (100 mL×3). The organic layers were combined and washed with waterand brine, then concentrated in vacuo. The residue was purified byreverse phase chromatography (CH₃CN/pure water=7:3) to afford(21E,23E,25E,26E,31R,32S,33R,34R,35S,38S,40S,42R,43R,52R)-42,52-dihydroxy-40-[(1R)-2-[(1S,2R,3R)-3-hydroxy-2-methoxy-cyclohexyl]-1-methyl-ethyl]-39-(3-hydroxypropoxy)-43-methoxy-31,32,33,34,44,45-hexamethyl-63,64-dioxa-53-azatricyclohexatriaconta-21,23,25(44),26(45)-tetraene-46,47,48,49,50-pentone(0.15 g, 29% yield) as a white solid. ESI-MS (EI⁺, m/z): 980.3 [M+Na]⁺.¹H NMR (500 MHz, CDCl₃) δ 6.58-5.84 (m, 4H), 5.72-4.83 (m, 4H),4.65-4.06 (m, 2H), 4.03-3.63 (m, 5H), 3.62-3.05 (m, 12H), 3.03-2.40 (m,6H), 2.42-1.91 (m, 7H), 1.89-1.56 (m, 17H), 1.53-1.27 (m, 6H), 1.25-0.76(m, 19H), 0.62 (m, 1H).

Example 12: Synthesis of(21E,23E,25E,26E,30R,31S,32R,33R,35R,37S,40S,41R,42R,51S)-41,51-dihydroxy-39-(2-hydroxyethoxy)-40-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-42-methoxy-30,31,32,33,43,44-hexamethyl-62,63-dioxa-52-azatricyclohexatriaconta-21,23,25(43),26(44)-tetraene-45,46,47,48,49-pentone(I-38)

Synthetic Scheme:

Procedures and Characterization:

p-toluenesulfonic acid hydrate (0.31 g, 1.64 mmol) was added to amixture of rapamycin (0.5 g, 0.547 mmol) and ethylene glycol (3 mL) inTHF (10 mL) at 10° C. The resulting mixture was stirred at 10° C. for 17hours then the reaction mixture was diluted with EtOAc (100 mL) andadjusted to pH 9 using saturated aqueous NaHCO₃ solution (about 50 mL).The organic layer was concentrated in vacuo then the residue waspurified by reverse phase chromatography (CH₃CN/pure water=5.5: 4.5).The solvent was then removed by lyophilization yielding(21E,23E,25E,26E,30R,31S,32R,33R,35R,37S,40S,41R,42R,51S)-41,51-dihydroxy-39-(2-hydroxyethoxy)-40-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-42-methoxy-30,31,32,33,43,44-hexamethyl-62,63-dioxa-52-azatricyclohexatriaconta-21,23,25(43),26(44)-tetraene-45,46,47,48,49-pentone(0.05 g, 9% yield) as a white solid. MS (EI⁺, m/z): 966.3 [M+Na]⁺. ¹HNMR (500 MHz, CDCl₃) δ 6.47-5.91 (m, 4H), 5.60-5.13 (m, 4H), 4.85-3.92(m, 3H), 3.88-3.68 (m, 4H), 3.60-3.53 (m, 1H), 3.46-3.29 (m, 10H),3.25-3.19 (m, 1H), 2.97-2.84 (m, 2H), 2.76-2.53 (m, 4H), 2.35-1.83 (m,8H), 1.80-1.64 (m, 12H), 1.53-1.16 (m, 9H), 1.14-0.82 (m, 18H),0.67-0.57 (m, 1H).

Example 13: Synthesis of(21E,23E,25E,26E,40R,41S,42R,43R,45R,47S,50S,51R,52R,61S)-51,61-dihydroxy-49-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-50-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-52-methoxy-40,41,42,43,53,54-hexamethyl-72,73-dioxa-62-azatricyclohexatriaconta-21,23,25(53),26(54)-tetraene-55,56,57,58,59-pentone(I-33)

Synthetic Scheme:

Procedures and Characterization:

p-toluenesulfonic acid hydrate (0.19 g, 1.02 mmol) was added to amixture of rapamycin (0.31 g, 0.34 mmol) and hexaethylene glycol (2 mL)in THF(6 mL) at 15° C. The resulting mixture was stirred at 15° C. for17 hours then the reaction mixture was diluted with EtOAc (200 mL) andadjusted to pH 9 using saturated aqueous NaHCO₃ solution (about 100 mL).The organic layer was concentrated in vacuo. The residue was purified byreverse phase chromatography (CH₃CN/pure water=3:2). The solvent wasthen removed by lyophilization.(21E,23E,25E,26E,40R,41S,42R,43R,45R,47S,50S,51R,52R,61S)-51,61-dihydroxy-49-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy] ethoxy] ethoxy] ethoxy]ethoxy]-50-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-52-methoxy-40,41,42,43,53,54-hexamethyl-72,73-dioxa-62-azatricyclohexatriaconta-21,23,25(53),26(54)-tetraene-55,56,57,58,59-pentone(I-33: 0.21 g, 51% yield) was obtained as a white solid. MS (EI⁺, m/z):1186.8 [M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ 6.39-5.94 (m, 4H), 5.55-5.14(m, 4H), 4.93-3.75 (m, 2H), 3.73-3.71 (m, 2H), 3.67-3.55 (m, 22H),3.48-3.44 (m, 1H), 3.41-3.16 (m, 12H), 2.98-2.86 (m, 2H), 2.75-2.69 (m,2H), 2.59-2.44 (m, 1H), 2.34-2.21 (m, 1H), 2.11-1.97 (m, 4H), 1.90 (s,3H), 1.78-1.54 (m, 13H), 1.50-1.12 (m, 9H), 1.08-0.83 (m, 18H),0.71-0.62 (m, 1H).

Example 14: Synthesis of(21E,23E,25E,26E,32R,33S,34R,35R,37R,39S,42S,43R,44R,53S)-43,53-dihydroxy-41-[2-(2-hydroxyethoxy)ethoxy]-42-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-44-methoxy-32,33,34,35,45,46-hexamethyl-64,65-dioxa-54-azatricyclohexatriaconta-21,23,25(45),26(46)-tetraene-47,48,49,50,51-pentone(I-18)

Synthetic Scheme:

Procedures and Characterization:

p-toluenesulfonic acid hydrate (0.187 g, 0.98 mmol) was added to amixture of rapamycin (0.3 g, 0.33 mmol) and diethylene glycol (2 mL) inTHF (6 mL) at 10° C. The resulting mixture was stirred at 10° C. for 17hours then the reaction mixture was diluted with EtOAc (100 mL) andadjusted to pH 9 using saturated aqueous NaHCO₃ solution (about 50 mL).The organic layer was concentrated under vacuum. The residue waspurified by reverse phase (CH₃CN/pure water=3:2). The solvents wereremoved by lyophilization, yielding(21E,23E,25E,26E,32R,33S,34R,35R,37R,39S,42S,43R,44R,53S)-43,53-dihydroxy-41-[2-(2-hydroxyethoxy)ethoxy]-42-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-44-methoxy-32,33,34,35,45,46-hexamethyl-64,65-dioxa-54-azatricyclohexatriaconta-21,23,25(45),26(46)-tetraene-47,48,49,50,51-pentone(I-18: 0.126 g, 37% yield) as a white solid. MS (EI⁺, m/z): 1010.7[M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ 6.40-5.95 (m, 4H), 5.55-5.14 (m, 4H),4.85-4.17 (m, 2H), 4.05-3.57 (m, 10H), 3.53-3.15 (m, 12H), 2.97-2.89 (m,2H), 2.76-2.48 (m, 4H), 2.36-1.84 (m, 7H), 1.79-1.56 (m, 14H), 1.49-1.14(m, 9H), 1.10-0.81 (m, 18H), 0.70-0.61 (m, 1H).

Example 15: Synthesis of(21E,23E,25E,26E,31R,32S,33R,34R,36R,38S,41S,42R,43R,53S)-42,53-dihydroxy-40-[3-hydroxy-2-(hydroxymethyl)propoxy]-41-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-43-methoxy-31,32,33,34,44,45-hexamethyl-65,66-dioxa-54-azatricyclohexatriaconta-21,23,25(44),26(45)-tetraene-46,47,48,49,50-pentone(I-31)

Synthetic Scheme:

Procedures and Characterization:

p-toluenesulfonic acid hydrate (0.31 g, 1.64 mmol) was added to amixture of rapamycin (0.5 g, 0.547 mmol) and 2-(hydroxymethyl)propane-1,3-diol (0.58 g, 5.47 mmol) in THF (10 mL) at 15° C. Theresulting mixture was stirred at 15° C. for 17 hours then the reactionmixture was diluted with EtOAc (100 mL) and adjusted to pH 9 usingsaturated aqueous NaHCO₃ solution (about 50 mL). The organic layer wasconcentrated under vacuum. The residue was purified by reverse phasechromatography (CH₃CN/pure water=3:2). The solvent was removed bylyophilization, yielding(21E,23E,25E,26E,31R,32S,33R,34R,36R,38S,41S,42R,43R,53S)-42,53-dihydroxy-40-[3-hydroxy-2-(hydroxymethyl)propoxy]-41-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-43-methoxy-31,32,33,34,44,45-hexamethyl-65,66-dioxa-54-azatricyclohexatriaconta-21,23,25(44),26(45)-tetraene-46,47,48,49,50-pentone(I-31: 0.054 g, 9% yield) as a white solid. MS (EI⁺, m/z): 1010.4[M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ 6.37-5.96 (m, 4H), 5.56-5.15 (m, 4H),4.86-4.17 (m, 2H), 3.85-3.47 (m, 10H), 2.96-2.91 (m, 2H), 2.75-2.58 (m,3H), 2.35-2.22 (m, 3H), 2.11-1.94 (m, 6H), 1.84-1.46 (m, 23H), 1.35-1.12(m, 9H), 1.11-0.88 (m, 18H), 0.67-0.65 (m, 1H).

Example 16: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethylsulfanyl)ethylsulfanyl]ethoxy]-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-21)

Synthetic Scheme:

Procedures and Characterization:

p-toluenesulfonic acid hydrate (0.62 g, 3.28 mmol) was added to amixture of rapamycin (1 g, 1.09 mmol) and2-[2-(2-hydroxyethylsulfanyl)ethylsulfanyl]ethanol (1.99 g, 10.94 mmol)in THF (20 mL) at 15° C. The resulting mixture was stirred at 15° C. for17 hours then the reaction mixture was diluted with EtOAc (100 mL) andadjusted to pH 9 using saturated aqueous NaHCO₃ solution (about 50 mL).The organic layer was concentrated under vacuum. The residue waspurified by reverse phase chromatography (CH₃CN/pure water=3:2). Thesolvents were removed by lyophilization yielding(21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethylsulfanyl)ethylsulfanyl]ethoxy]-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-21: 0.15 g, 0.133 mmol, 12% yield) as a yellow solid. MS (EI⁺, m/z):1086.4 [M+Na]⁺.

¹H NMR (500 MHz, CDCl₃) δ 6.39-5.95 (m, 4H), 5.54-5.19 (m, 4H),4.81-4.17 (m, 2H), 3.96-3.73 (m, 4H), 3.59-3.14 (m, 12H), 2.96-2.55 (m,14H), 2.35-1.87 (m, 6H), 1.81-1.59 (m, 13H), 1.53-1.13 (m, 11H),1.16-0.84 (m, 18H), 0.71-0.63 (m, 1H).

Example 17: Synthesis of Synthesis of(21E,23E,25E,26E,42R,43S,44R,45R,47S,49S,51S,52S,53R,54R,63R)-53,63-dihydroxy-51-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-52-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-54-methoxy-42,43,44,45,55,56-hexamethyl-74,75-dioxa-64-azatricyclohexatriaconta-21,23,25(55),26(56)-tetraene-57,58,59,60,61-pentone(I-26)

Synthetic Scheme:

Procedures and Characterization:

p-toluenesulfonic acid hydrate (0.52 g, 2.73 mmol) was added slowly to asolution of rapamycin (0.5 g, 0.55 mmol) and2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol(3.57 g, 10.94 mmol) in THF (10 mL). The resultant solution was stirredat 20° C. for 17 hours and then concentrated. The residue was purifiedby reverse phase chromatography (CH₃CN/pure water=7:3) to obtain(21E,23E,25E,26E,42R,43S,44R,45R,47S,49S,51S,52S,53R,54R,63R)-53,63-dihydroxy-51-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-52-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-54-methoxy-42,43,44,45,55,56-hexamethyl-74,75-dioxa-64-azatricyclohexatriaconta-21,23,25(55),26(56)-tetraene-57,58,59,60,61-pentone(I-26: 0.16 g, 24% yield) as a white solid. MS (EI⁺, m/z):1230.6[M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ 6.46-5.74 (m, 4H), 5.61-4.74(m, 4H), 4.05-4.5 (m, 2H), 4.02-3.51 (m, 35H), 3.43-3.16 (m, 14H),2.99-2.42 (m, 8H), 2.4-1.6 (m, 7H), 1.61-1.1 (m, 12H), 1.13-0.79 (m,18H), 0.74-0.61 (m, 1H).

Example 19: Synthesis of(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-42-[R-2-(2-methoxyethoxy)ethoxy]-33,34,35,36,46,47-hexamethyl-64,65-dioxa-55-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraene-48,49,50,51,52-pentone(I-119) and(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-42-[S-2-(2-methoxyethoxy)ethoxy]-33,34,35,36,46,47-hexamethyl-64,65-dioxa-55-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraene-48,49,50,51,52-pentone(I-120)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-42-[2-(2-methoxyethoxy)ethoxy]-33,34,35,36,46,47-hexamethyl-64,65-dioxa-55-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraene-48,49,50,51,52-pentone

To a mixture of rapamycin (2 g, 2.19 mmol), 2-(2-methoxyethoxy)ethanol(4 mL) in THF (30 mL) was added HND-8 (240 mg, 2.19 mmol) and thereaction stirred at 50° C. for 4 h under N₂, then filtered andconcentrated. The resulting crude product was purified via reverse phasechromatography (C18, CH₃CN: H₂O=3:1) to provide(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-42-[2-(2-methoxyethoxy)ethoxy]-33,34,35,36,46,47-hexamethyl-64,65-dioxa-55-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraene-48,49,50,51,52-pentone(0.6 g, 27% yield) as a light yellow solid. ESI-MS (EI⁺, m/z): 1024.3[M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ 6.56-5.82 (m, 4H), 5.47 (ddd, J=37.4,17.7, 9.4 Hz, 2H), 5.31-5.06 (m, 2H), 4.82-4.50 (m, 1H), 4.32-3.94 (m,2H), 3.92-3.71 (m, 2H), 3.70-3.44 (m, 8H), 3.43-3.26 (m, 12H), 3.20 (dd,J=27.0, 16.2 Hz, 1H), 3.00-2.22 (m, 7H), 2.18-1.56 (m, 19H), 1.54-1.25(m, 7H), 1.24-0.81 (m, 19H), 0.67 (dd, J=23.8, 11.9 Hz, 1H).

Step 2: Synthesis of(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-42-[R-2-(2-methoxyethoxy)ethoxy]-33,34,35,36,46,47-hexamethyl-64,65-dioxa-55-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraene-48,49,50,51,52-pentone(I-119) and(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-42-[S-2-(2-methoxyethoxy)ethoxy]-33,34,35,36,46,47-hexamethyl-64,65-dioxa-55-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraene-48,49,50,51,52-pentone(I-120)

(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-42-[2-(2-methoxyethoxy)ethoxy]-33,34,35,36,46,47-hexamethyl-64,65-dioxa-55-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraene-48,49,50,51,52-pentone(0.095 g, 0.095 mmol) was purified via prep chiral HPLC to provide(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-42-[R-2-(2-methoxyethoxy)ethoxy]-33,34,35,36,46,47-hexamethyl-64,65-dioxa-55-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraene-48,49,50,51,52-pentone(I-119: 13.2 mg, 14% yield) and(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-42-[S-2-(2-methoxyethoxy)ethoxy]-33,34,35,36,46,47-hexamethyl-64,65-dioxa-55-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraene-48,49,50,51,52-pentone(I-120: 18.1 mg, 19% yield), both as white solids.

The chiral separation method is listed below:

Instrument: Gilson-281

Column: CHIRALPAK IC 20×250 mm, 10 um (Daicel)

Column temperature: 35° C.

Mobile phase: n-Hexane:Ethanol=60:40

Flow rate: 50 ml/min

Detection wavelength: 214 nm

Cycle time: 18 min

Sample solution: 95 mg dissolved in 7m Methanol

Injection volume: 0.5 ml (loading: 7.1 mg/injection)

I-119: ESI-MS (EI⁺, m/z): 1024.4 [M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ6.42-5.92 (m, 4H), 5.75-5.05 (m, 4H), 4.49 (s, 1H), 4.28 (s, 1H), 4.15(d, J=10.9 Hz, 1H), 3.99 (t, J=13.3 Hz, 1H), 3.88-3.73 (m, 1H),3.69-3.46 (m, 8H), 3.45-3.29 (m, 11H), 3.22 (dd, J=10.1, 6.5 Hz, 2H),2.99-2.77 (m, 3H), 2.68 (dt, J=28.5, 11.1 Hz, 3H), 2.61-2.22 (m, 4H),2.05 (ddd, J=21.5, 15.3, 7.5 Hz, 5H), 1.88-1.65 (m, 12H), 1.53-1.30 (m,7H), 1.17-0.78 (m, 19H), 0.73-0.57 (m, 1H).

I-120: ESI-MS (EI⁺, m/z): 1024.3 [M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ6.43-6.18 (m, 2H), 6.13 (dd, J=15.0, 10.0 Hz, 1H), 5.91 (dd, J=31.6,10.6 Hz, 1H), 5.56-5.36 (m, 2H), 5.27 (d, J=4.9 Hz, 1H), 5.16 (dt,J=18.2, 9.1 Hz, 1H), 4.78 (s, 1H), 4.20 (dd, J=21.1, 11.1 Hz, 1H),3.96-3.71 (m, 3H), 3.70-3.43 (m, 9H), 3.42-3.26 (m, 14H), 2.98-2.87 (m,1H), 2.77-2.62 (m, 3H), 2.58 (dd, J=16.9, 6.3 Hz, 1H), 2.34 (d, J=13.4Hz, 2H), 2.15-1.84 (m, 5H), 1.83-1.64 (m, 7H), 1.34 (dddd, J=22.7, 19.4,18.1, 9.1 Hz, 12H), 1.16-0.80 (m, 19H), 0.66 (dt, J=16.8, 8.4 Hz, 1H).

Example 20: Synthesis of(23E,25E,27E,28E,32R,33S,34R,35R,36S,39S,41S,45R,46R,55R)-45,55-dihydroxy-41-[(1R)-2-[(1S,2R,3R)-3-hydroxy-2-methoxy-cyclohexyl]-1-methyl-ethyl]-40-[[(2S,5S)-5-(hydroxymethyl)-1,4-dioxan-2-yl]methoxy]-46-methoxy-32,33,34,35,47,48-hexamethyl-68,69-dioxa-56-azatricyclohexatriaconta-23,25,27(47),28(48)-tetraene-49,50,51,52,53-pentone(I-118)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of(S)-1-(benzyloxy)-3-chloropropan-2-ol

A solution of (2R)-2-(chloromethyl)oxirane (1 g, 10.81 mmol) andphenylmethanol (2.34 g, 21.62 mmol) in DCE (10 mL) was stirred at rt for1 h then cooled to 0° C., boron trifluoride etherate (76.7 mg, 0.54mmol) was added slowly. The reaction mixture was allowed to warm to rt,stirred overnight then refluxed for 2 h. Upon cooling, the reactionmixture was diluted with 10% NaHCO₃ aqueous solution and extracted withEtOAc (100 mL×3). The combined organic layers were dried over anhydrousNa₂SO₄, filtered, and concentrated. The resulting residue was purifiedvia silica gel chromatography (PE: EtOAc=10: 1) to provide the desiredproduct (1.8 g, 86% yield) as a colorless oil. ESI-MS (EI⁺, m/z): 218.1[M+H₂O]⁺. ¹H NMR (500 MHz, CDCl₃) δ 7.36-7.27 (m, 5H), 4.64 (s, 2H),3.99-3.95 (m, 1H), 3.64-3.55 (m, 4H), 2.75 (d, J=6.0 Hz, 1H).

Step 2: Synthesis of(S)-2-((S)-1-(benzyloxy)-3-chloropropan-2-yloxy)-3-hydroxypropyl4-methylbenzenesulfonate

A solution of (2S)-1-benzyloxy-3-chloro-propan-2-ol (6 g, 29.9 mmol) and[(2S)-oxiran-2-yl]methyl 4-methylbenzenesulfonate (6.83 g, 29.9 mmol) inDCE (100 mL) was stirred at rt for 1 h then cooled to 0° C. and borontrifluoride etherate (254.6 mg, 1.79 mmol) was added slowly. Thereaction was warmed to rt, stirred at rt overnight, refluxed for 2h thencooled to rt. EtOAc (100 mL) and water (50 mL) were added. The layerswere separated and the aqueous layer further extracted with EtOAc (100mL×2) The combined organic layers were then washed with water (2×50 mL)and brine (50 mL), dried over anhydrous Na₂SO₄, filtered andconcentrated under vacuum. The resulting residue was purified by silicagel chromatography (8% EtOAc in PE) to afford[(2S)-2-[(1S)-1-(benzyloxymethyl)-2-chloro-ethoxy]-3-hydroxy-propyl]4-methylbenzenesulfonate (4.4 g, 34% yield) as a colorless oil. ESI-MS(EI⁺, m/z): 429.1 [M+H]⁺.

Step 3: Synthesis of((2R,5R)-5-(benzyloxymethyl)-1,4-dioxan-2-yl)methanol

A solution of[(2S)-2-[(1S)-1-(benzyloxymethyl)-2-chloro-ethoxy]-3-hydroxy-propyl]4-methylbenzenesulfonate (0.78 g, 1.82 mmol), NaOH (0.22 g, 5.46 mmol)in H₂O (10 mL) was stirred at room temperature for 2.5 h, then heated at90° C. for 4 h, cooled to rt and stirred overnight, then heated at 90°C. for another 2 h. The reaction mixture was acidified with 1N HClaqueous solution and extracted with DCM (100 mL×3). The combined organiclayers were washed with NaHCO₃ aqueous solution, dried over anhydrousNa₂SO₄, filtered and concentrated to obtain[(2R,5R)-5-(benzyloxymethyl)-1,4-dioxan-2-yl]methanol (4.4 g, 41% yield)as a colorless oil. This material was used without further purification.ESI-MS (EI⁺, m/z): 239.1 [M+H]⁺.

Step 4: Synthesis of ((2R,5R)-1,4-dioxane-2,5-diyl)dimethanol

To a solution of [(2R,5R)-5-(benzyloxymethyl)-1,4-dioxan-2-yl]methanol(2.4 g, 10 mmol) in MeOH (30 mL) was added Pd/C (0.86 g). The mixturewas allowed to stir at rt overnight under a hydrogen balloon thenfiltered through a short celite plug, washing with ethanol. The combinedorganic washes were concentrated under reduced pressure to provide(2R,5R)-1,4-dioxane-2,5-diyl)dimethanol (1.2 g, 74% yield) as an oil.ESI-MS (EI⁺, m/z): 149.2 [M+H]⁺.

Step 5: Synthesis of(23E,25E,27E,28E,32R,33S,34R,35R,36S,39S,41S,45R,46R,55R)-45,55-dihydroxy-41-[(1R)-2-[(1S,2R,3R)-3-hydroxy-2-methoxy-cyclohexyl]-1-methyl-ethyl]-40-[[(2S,5S)-5-(hydroxymethyl)-1,4-dioxan-2-yl]methoxy]-46-methoxy-32,33,34,35,47,48-hexamethyl-68,69-dioxa-56-azatricyclohexatriaconta-23,25,27(47),28(48)-tetraene-49,50,51,52,53-pentone(I-118)

To a solution of rapamycin (0.5 g, 0.547 mmol) and4-methylbenzenesulfonic acid hydrate (0.471 g, 2.73 mmol) in THF (15 mL)was added [(2R,5R)-5-(hydroxymethyl)-1,4-dioxan-2-yl]methanol (0.81 g,5.47 mmol) at 25° C. After stirring at rt for 2 h, the reaction wasquenched with cold NaHCO₃ aqueous solution and extracted with EtOAc (50mL×2). The combined organic layers were then dried over anhydrousNa₂SO₄, filtered and concentrated. The residue was purified viareverse-phase chromatography (C18, CH₃CN:H₂O=65:35) followed via silicagel chromatography (hexane:DCM:EtOAc:MeOH=8:8:3:1) to obtain(23E,25E,27E,28E,32R,33S,34R,35R,36S,39S,41S,45R,46R,55R)-45,55-dihydroxy-41-[(1R)-2-[(1S,2R,3R)-3-hydroxy-2-methoxy-cyclohexyl]-1-methyl-ethyl]-40-[[(2S,5S)-5-(hydroxymethyl)-1,4-dioxan-2-yl]methoxy]-46-methoxy-32,33,34,35,47,48-hexamethyl-68,69-dioxa-56-azatricyclohexatriaconta-23,25,27(47),28(48)-tetraene-49,50,51,52,53-pentone(I-118: 80 mg, 14% yield) as a white solid. ESI-MS (EI⁺, m/z): 1052.9[M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ 6.46-5.86 (m, 4H), 5.69-5.06 (m, 4H),4.16 (ddd, J=56.1, 52.0, 39.5 Hz, 3H), 3.94-3.49 (m, 10H), 3.48-3.11 (m,12H), 3.08-2.46 (m, 7H), 2.40-1.93 (m, 7H), 1.73 (dd, J=16.9, 10.5 Hz,13H), 1.52-1.17 (m, 8H), 1.16-0.79 (m, 18H), 0.65 (d, J=17.5 Hz, 1H).

Example 21: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43R,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-(2-hydroxyethoxy)ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-116) and(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-(2-hydroxyethoxy)ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-117)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-(2-hydroxyethoxy)ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone

A solution of everolimus (1 g, 1.04 mmol) in THF (15 mL) was degassedwith N₂. p-toluenesulfonic acid (0.895 g, 5.20 mmol) was added at 0° C.followed by 2-(2-hydroxyethoxy)ethanol (2.8 mL). The resulting mixturewas stirred at 0° C. for 0.5 h under N₂, then at 25° C. for 3 h. Thereaction was poured into sat.NaHCO₃(40 mL), extracted with EtOAc (30mL), washed with water (30 mL×2) and brine (40 mL) then dried overanhydrous Na₂SO₄, filtered and concentrated. The resulting residue waspurified by normal phase silica gel chromatography (MeOH: DCM=1:15) thenreverse-phase chromatography (C18, CH₃CN: H₂O=7: 3) to obtain(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-(2-hydroxyethoxy)ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(0.43 g, 40% yield) as a light yellow solid. ESI-MS (EI⁺, m/z): 1053.9[M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ 6.47-5.85 (m, 4H), 5.40 (ddd, J=99.7,51.9, 29.1 Hz, 4H), 4.80 (d, J=22.0 Hz, 1H), 4.23 (d, J=42.5 Hz, 1H),4.05-3.54 (m, 13H), 3.52-3.01 (m, 14H), 2.67 (ddd, J=46.8, 27.3, 6.8 Hz,4H), 2.17 (dd, J=82.0, 45.9 Hz, 6H), 1.70 (dt, J=21.0, 15.8 Hz, 12H),1.34 (dd, J=105.2, 26.3 Hz, 11H), 1.15-0.79 (m, 18H), 0.76-0.64 (m, 1H).

Step 2: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43R,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-(2-hydroxyethoxy)ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-116) and(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-(2-hydroxyethoxy)ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-117)

90 mg of(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-(2-hydroxyethoxy)ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentonewas purified via prep chiral HPLC and the resulting epimers wereobtained:(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43R,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-(2-hydroxyethoxy)ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-116: 14 mg, 16% yield) and(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-(2-hydroxyethoxy)ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-117: 15 mg, 17% yield), both as white solids.

The chiral separation method is listed below:

Column: CHIRALPAK IC

Column size: 5.0 cm I.D.×25 cm L

Solution concentration: 11.5 mg/ml

Injection: 10 ml

Mobile phase: Hexane/EtOH=50/50(V/V)

Flow rate: 60 ml/min

Wave length: UV 254 nm

Temperature: 35° C.

I-116: ESI-MS (EI⁺, m/z): 1054.0 [M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ6.42-5.90 (m, 4H), 5.79 (ddd, J=51.6, 30.9, 16.6 Hz, 1H), 5.54-5.08 (m,4H), 5.03-4.88 (m, 1H), 4.74 (d, J=61.6 Hz, 1H), 4.28 (dd, J=57.6, 29.0Hz, 2H), 3.99 (dd, J=26.5, 6.0 Hz, 1H), 3.89-3.55 (m, 12H), 3.54-2.96(m, 15H), 2.87-2.47 (m, 4H), 2.38-1.92 (m, 8H), 1.86-1.67 (m, 11H),1.51-1.30 (m, 6H), 1.14-0.80 (m, 18H), 0.76-0.64 (m, 1H).

I-117: ESI-MS (EI⁺, m/z): 1053.9 [M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ6.41-6.22 (m, 2H), 6.13 (dd, J=15.1, 10.1 Hz, 1H), 5.94 (dd, J=22.3,10.8 Hz, 1H), 5.52 (dt, J=18.4, 9.2 Hz, 1H), 5.41 (d, J=9.9 Hz, 1H),5.27 (d, J=5.3 Hz, 1H), 5.12 (dt, J=46.3, 5.6 Hz, 1H), 4.83 (s, 1H),4.23-4.14 (m, 1H), 3.91-3.52 (m, 15H), 3.49-3.25 (m, 12H), 3.23-3.03 (m,3H), 2.94-2.80 (m, 1H), 2.65 (ddd, J=23.4, 16.9, 6.0 Hz, 3H), 2.39-2.15(m, 2H), 2.16-1.85 (m, 5H), 1.82-1.64 (m, 10H), 1.47 (dd, J=26.8, 15.9Hz, 5H), 1.38-1.16 (m, 6H), 1.10 (d, J=6.8 Hz, 3H), 1.07-1.03 (m, 3H),1.00 (t, J=6.8 Hz, 3H), 0.94 (d, J=6.6 Hz, 3H), 0.88 (ddd, J=34.0, 16.9,5.0 Hz, 6H), 0.71 (dd, J=23.9, 11.8 Hz, 1H).

Example 22: Synthesis of(21E,23E,25E,26E,38R,39S,40R,41R,43S,45S,47R,48S,49R,50R,59R)-49,59-dihydroxy-47-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-48-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-50-methoxy-38,39,40,41,51,52-hexamethyl-70,71-dioxa-60-azatricyclohexatriaconta-21,23,25(51),26(52)-tetraene-53,54,55,56,57-pentone(I-114) and(21E,23E,25E,26E,38R,39S,40R,41R,43S,45S,47S,48S,49R,50R,59R)-49,59-dihydroxy-47-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-48-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-50-methoxy-38,39,40,41,51,52-hexamethyl-70,71-dioxa-60-azatricyclohexatriaconta-21,23,25(51),26(52)-tetraene-53,54,55,56,57-pentone(I-115)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of(21E,23E,25E,26E,38R,39S,40R,41R,43S,45S,48S,49R,50R,59R)-49,59-dihydroxy-47-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-48-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-50-methoxy-38,39,40,41,51,52-hexamethyl-70,71-dioxa-60-azatricyclohexatriaconta-21,23,25(51),26(52)-tetraene-53,54,55,56,57-pentone

A mixture of everolimus (1 g, 1.04 mmol),2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethanol (4.05 g, 20.87 mmol) andp-toluenesulfonic acid (0.898 g, 5.22 mmol) in THF (20 mL) was stirredat 20° C. for 2 h. The mixture was then poured into cold sat.NaHCO₃(30mL), extracted with EtOAc (50 mL×3) and the combined organic layerswashed with water and brine then dried over anhydrous Na₂SO₄, filteredand concentrated. The resulting residue was purified by reversed phasechromatography (C18, CH₃CN: H₂O=6:4) to afford(21E,23E,25E,26E,38R,39S,40R,41R,43S,45S,48S,49R,50R,59R)-49,59-dihydroxy-47-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-48-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-50-methoxy-38,39,40,41,51,52-hexamethyl-70,71-dioxa-60-azatricyclohexatriaconta-21,23,25(51),26(52)-tetraene-53,54,55,56,57-pentone(0.25 g, 21% yield) as white solid. ESI-MS (EI⁺, m/z): 1142.4 [M+Na]⁺.¹H NMR (400 MHz, CDCl₃) δ 6.41-5.85 (m, 4H), 5.78 (s, 1H), 5.60-4.98 (m,4H), 4.22 (t, J=27.1 Hz, 1H), 3.97 (dd, J=17.7, 6.4 Hz, 1H), 3.87-3.54(m, 21H), 3.51-3.03 (m, 15H), 2.72-2.45 (m, 3H), 2.28 (s, 6H), 2.16-1.96(m, 4H), 1.89-1.56 (m, 10H), 1.51-1.17 (m, 8H), 1.17-0.81 (m, 18H),0.78-0.62 (m, 1H).

Step 2: Synthesis of(21E,23E,25E,26E,38R,39S,40R,41R,43S,45S,47R,48S,49R,50R,59R)-49,59-dihydroxy-47-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-48-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-50-methoxy-38,39,40,41,51,52-hexamethyl-70,71-dioxa-60-azatricyclohexatriaconta-21,23,25(51),26(52)-tetraene-53,54,55,56,57-pentone(I-114) and(21E,23E,25E,26E,38R,39S,40R,41R,43S,45S,47S,48S,49R,50R,59R)-49,59-dihydroxy-47-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-48-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-50-methoxy-38,39,40,41,51,52-hexamethyl-70,71-dioxa-60-azatricyclohexatriaconta-21,23,25(51),26(52)-tetraene-53,54,55,56,57-pentone(I-115)

1.5 g of(21E,23E,25E,26E,38R,39S,40R,41R,43S,45S,48S,49R,50R,59R)-49,59-dihydroxy-47-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-48-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-50-methoxy-38,39,40,41,51,52-hexamethyl-70,71-dioxa-60-azatricyclohexatriaconta-21,23,25(51),26(52)-tetraene-53,54,55,56,57-pentonewas purified via prep chiral HPLC and the resulting epimers purified viasilica gel chromatography (hexane:DCM:EtOAc:MeOH=8:8:3:1.2) to obtain(21E,23E,25E,26E,38R,39S,40R,41R,43S,45S,47R,48S,49R,50R,59R)-49,59-dihydroxy-47-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-48-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-50-methoxy-38,39,40,41,51,52-hexamethyl-70,71-dioxa-60-azatricyclohexatriaconta-21,23,25(51),26(52)-tetraene-53,54,55,56,57-pentone(I-114: 300 mg, 20% yield) and(21E,23E,25E,26E,38R,39S,40R,41R,43S,45S,47S,48S,49R,50R,59R)-49,59-dihydroxy-47-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-48-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-50-methoxy-38,39,40,41,51,52-hexamethyl-70,71-dioxa-60-azatricyclohexatriaconta-21,23,25(51),26(52)-tetraene-53,54,55,56,57-pentone(I-115: 563 mg, 38% yield) as a white solids.

Chiral Analysis Conditions:

Column: CHIRALPAK IC-3(IC30CE-NJ008)

Column size: 0.46 cm I.D.×15 cm L

Injection: 20.0 ul

Mobile phase: Hexane/EtOH=60/40(V/V)

Flow rate: 1.0 ml/min

Wave length: UV 254 nm

Temperature: 35° C.

HPLC equipment: Shimadzu LC-20AT

I-114: ESI-MS (EI⁺, m/z): 1142.5 [M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ6.42-5.91 (m, 4H), 5.60-5.08 (m, 4H), 4.19 (dd, J=43.8, 32.6 Hz, 1H),3.96 (dd, J=27.1, 6.3 Hz, 1H), 3.85-3.54 (m, 21H), 3.53-3.01 (m, 12H),2.93-2.80 (m, 1H), 2.75-2.45 (m, 3H), 2.30 (d, J=12.1 Hz, 1H), 2.03 (dd,J=37.0, 32.8 Hz, 13H), 1.84-1.69 (m, 12H), 1.50-1.18 (m, 5H), 1.16-0.82(m, 18H), 0.71 (dt, J=23.8, 12.1 Hz, 1H).

I-115: ESI-MS (EI⁺, m/z): 1142.4 [M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ 6.36(dd, J=14.7, 10.9 Hz, 1H), 6.23 (dd, J=14.7, 10.6 Hz, 1H), 6.10 (dd,J=15.0, 10.5 Hz, 1H), 5.92 (dd, J=45.2, 10.5 Hz, 1H), 5.61 (s, 1H), 5.43(dd, J=15.6, 9.6 Hz, 2H), 5.21 (d, J=5.4 Hz, 1H), 5.10 (dd, J=9.9, 5.9Hz, 1H), 4.20 (d, J=4.5 Hz, 1H), 4.10-3.95 (m, 1H), 3.84 (d, J=5.0 Hz,1H), 3.80-3.49 (m, 21H), 3.48-3.13 (m, 12H), 3.11-3.01 (m, 1H),2.73-2.50 (m, 3H), 2.34-2.19 (m, 2H), 2.01 (ddd, J=62.0, 34.6, 28.2 Hz,12H), 1.80-1.54 (m, 10H), 1.51-1.37 (m, 5H), 1.35-1.12 (m, 6H), 1.05(dd, J=6.4, 5.0 Hz, 6H), 0.97 (d, J=6.5 Hz, 3H), 0.93 (d, J=6.6 Hz, 3H),0.91-0.82 (m, 6H), 0.74-0.67 (m, 1H).

Example 23: Synthesis of(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-42-(5-hydroxypentoxy)-45-methoxy-33,34,35,36,46,47-hexamethyl-65,66-dioxa-55-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone(I-113)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-42-(5-hydroxypentoxy)-45-methoxy-33,34,35,36,46,47-hexamethyl-65,66-dioxa-55-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone(I-113)

To a solution of rapamycin (0.5 g, 0.547 mmol) in THF (10 mL) was added4-methylbenzenesulfonic acid hydrate (0.52 g, 2.73 mmol) andpentane-1,5-diol (3 mL). The resulting solution was stirred at rt for 2h then poured into cold NaHCO₃ aqueous solution and extracted withEtOAc. The organic layer was concentrated and purified by reverse-phasechromatography (C18, CH₃CN:H₂O from 10% to 72% yield) to afford(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-42-(5-hydroxypentoxy)-45-methoxy-33,34,35,36,46,47-hexamethyl-65,66-dioxa-55-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone(I-113: 150 mg, 28% yield) as a white solid. ESI-MS (EI⁺, m/z): 1008.0[M+Na]⁺. ¹HNMR (500 MHz, CDCl₃) δ 6.42-5.82 (m, 4H), 5.58-5.37 (m, 2H),5.32-5.02 (m, 2H), 4.78 (t, J=25.9 Hz, 1H), 4.31-4.08 (m, 1H), 4.00-3.53(m, 5H), 3.53-3.05 (m, 12H), 2.99-2.80 (m, 2H), 2.77-2.51 (m, 3H),2.48-2.23 (m, 2H), 2.15-1.89 (m, 4H), 1.89-1.16 (m, 32H), 1.15-0.78 (m,18H), 0.65 (dt, J=24.1, 12.0 Hz, 1H).

Example 24: Synthesis of(28E,30E,32E,33E,36R,37S,38R,39R,41S,43S,45R,46S,48R,49R,58R)-48,58-dihydroxy-46-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-49-methoxy-36,37,38,39,50,51-hexamethyl-45-(1,4,7,10-tetraoxacyclododec-2-ylmethoxy)-72,73-dioxa-59-azatricyclohexatriaconta-28,30,32(50),33(51)-tetraene-52,53,54,55,56-pentone(I-111) and(28E,30E,32E,33E,36R,37S,38R,39R,41S,43S,45S,46S,48R,49R,58R)-48,58-dihydroxy-46-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-49-methoxy-36,37,38,39,50,51-hexamethyl-45-(1,4,7,10-tetraoxacyclododec-2-ylmethoxy)-72,73-dioxa-59-azatricyclohexatriaconta-28,30,32(50),33(51)-tetraene-52,53,54,55,56-pentone(I-112)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of(28E,30E,32E,33E,36R,37S,38R,39R,41S,43S,46S,48R,49R,58R)-48,58-dihydroxy-46-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-49-methoxy-36,37,38,39,50,51-hexamethyl-45-(1,4,7,10-tetraoxacyclododec-2-ylmethoxy)-72,73-dioxa-59-azatricyclohexatriaconta-28,30,32(50),33(51)-tetraene-52,53,54,55,56-pentone

To a solution of rapamycin (0.5 g, 0.547 mmol) and4-methylbenzenesulfonic acid hydrate (0.471 g, 2.73 mmol) in THF (15 mL)was added 1,4,7,10-tetraoxacyclododec-2-ylmethanol (2.25 g, 10.9 mmol)at 25° C. The resulting mixture was stirred at rt for 2 h then pouredinto ice cold NaHCO₃ aqueous solution and extracted with EtOAc. Theorganic layer was then then dried over anhydrous Na₂SO₄, filtered andconcentrated. The resulting residue was purified by reverse-phasechromatography (C18, CH₃CN:H₂O=7:3) to obtain(28E,30E,32E,33E,36R,37S,38R,39R,41S,43S,46S,48R,49R,58R)-48,58-dihydroxy-46-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-49-methoxy-36,37,38,39,50,51-hexamethyl-45-(1,4,7,10-tetraoxacyclododec-2-ylmethoxy)-72,73-dioxa-59-azatricyclohexatriaconta-28,30,32(50),33(51)-tetraene-52,53,54,55,56-pentone(70 mg, 11% yield) as a white solid. ESI-MS (EI⁺, m/z): 1110.5 [M+Na]⁺.¹HNMR (500 MHz, CDCl₃) δ 6.41-5.82 (m, 4H), 5.54-5.04 (m, 4H), 4.72 (d,J=22.1 Hz, 1H), 4.35-4.09 (m, 1H), 3.92-3.51 (m, 18H), 3.48-3.03 (m,14H), 2.99-2.51 (m, 5H), 2.34 (d, J=13.4 Hz, 1H), 2.04 (d, J=62.6 Hz,4H), 1.72 (ddd, J=43.8, 30.6, 28.9 Hz, 12H), 1.53-1.17 (m, 10H),1.14-0.81 (m, 18H), 0.70-0.62 (m, 1H).

Step 2: Synthesis of(28E,30E,32E,33E,36R,37S,38R,39R,41S,43S,45S,46S,48R,49R,58R)-48,58-dihydroxy-46-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-49-methoxy-36,37,38,39,50,51-hexamethyl-45-(1,4,7,10-tetraoxacyclododec-2-ylmethoxy)-72,73-dioxa-59-azatricyclohexatriaconta-28,30,32(50),33(51)-tetraene-52,53,54,55,56-pentone(I-112) and(28E,30E,32E,33E,36R,37S,38R,39R,41S,43S,45R,46S,48R,49R,58R)-48,58-dihydroxy-46-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-49-methoxy-36,37,38,39,50,51-hexamethyl-45-(1,4,7,10-tetraoxacyclododec-2-ylmethoxy)-72,73-dioxa-59-azatricyclohexatriaconta-28,30,32(50),33(51)-tetraene-52,53,54,55,56-pentone(I-11)

(28E,30E,32E,33E,36R,37S,38R,39R,41S,43S,46S,48R,49R,58R)-48,58-dihydroxy-46-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-49-methoxy-36,37,38,39,50,51-hexamethyl-45-(1,4,7,10-tetraoxacyclododec-2-ylmethoxy)-72,73-dioxa-59-azatricyclohexatriaconta-28,30,32(50),33(51)-tetraene-52,53,54,55,56-pentone(170 mg) was purified via prep chiral HPLC and the resulting epimerspurified via silica gel chromatography (hexane:DCM:EtOAc:MeOH=3:3:1:0.5)to obtain(28E,30E,32E,33E,36R,37S,38R,39R,41S,43S,45S,46S,48R,49R,58R)-48,58-dihydroxy-46-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-49-methoxy-36,37,38,39,50,51-hexamethyl-45-(1,4,7,10-tetraoxacyclododec-2-ylmethoxy)-72,73-dioxa-59-azatricyclohexatriaconta-28,30,32(50),33(51)-tetraene-52,53,54,55,56-pentone(I-112: 55 mg, 32% yield) and(28E,30E,32E,33E,36R,37S,38R,39R,41S,43S,45R,46S,48R,49R,58R)-48,58-dihydroxy-46-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-49-methoxy-36,37,38,39,50,51-hexamethyl-45-(1,4,7,10-tetraoxacyclododec-2-ylmethoxy)-72,73-dioxa-59-azatricyclohexatriaconta-28,30,32(50),33(51)-tetraene-52,53,54,55,56-pentone(I-111: 11 mg, 6% yield), both as white solids.

Chiral Analysis Method:

Column: CHIRALPAK IC(IC00CE-OL002)

Column size: 0.46 cm I.D.×25 cm L

Injection: 100.0 ul

Mobile phase: Hexane/EtOH=60/40 (V/V)

Flow rate: 1.0 ml/min

Wave length: UV 254 nm

Temperature: 35° C.

HPLC equipment: Shimadzu LC-20AT CP-HPLC-06

I-112: ESI-MS (EI⁺, m/z): 1109.9 [M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ6.40-6.03 (m, 3H), 5.90 (dd, J=37.1, 9.8 Hz, 1H), 5.54-5.06 (m, 4H),4.71 (d, J=22.4 Hz, 1H), 4.20 (t, J=20.9 Hz, 1H), 3.93-3.52 (m, 17H),3.50-3.24 (m, 11H), 3.14-3.02 (m, 1H), 2.93 (dt, J=31.2, 12.0 Hz, 1H),2.63 (tdd, J=17.0, 14.2, 5.5 Hz, 3H), 2.37-2.18 (m, 2H), 1.94 (ddd,J=31.3, 24.3, 22.1 Hz, 5H), 1.71 (dt, J=22.5, 10.1 Hz, 11H), 1.51-1.17(m, 13H), 1.15-0.81 (m, 18H), 0.67 (dd, J=23.7, 11.9 Hz, 1H).

I-111: ¹H NMR (500 MHz, CDCl₃) δ 6.46-5.78 (m, 4H), 5.75-5.14 (m, 4H),4.58 (d, J=31.9 Hz, 1H), 4.21 (d, J=66.8 Hz, 1H), 3.87-3.47 (m, 16H),3.43-3.14 (m, 10H), 2.94 (s, 1H), 2.80-2.54 (m, 3H), 2.26 (ddd, J=110.3,80.5, 42.6 Hz, 6H), 1.81-1.49 (m, 18H), 1.46-1.25 (m, 10H), 1.18-0.77(m, 18H), 0.72-0.61 (m, 1H).

Example 25: Synthesis of(21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,45R,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone(I-109) and(21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,45S,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone(I-110)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of(21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone

A mixture of everolimus (0.5 g, 0.522 mmol),2-[2-(2-hydroxyethoxy)ethoxy]ethanol (3.92 g, 26.09 mmol) andp-toluenesulfonic acid monohydrate (0.45 g, 2.61 mmol) in THF (10 mL)was stirred at 20° C. for 2 h. The mixture was then poured into ice coldsat. NaHCO₃(30 mL) and extracted with EtOAc (50 mL×3). The combinedorganic layers were washed with water and brine then dried overanhydrous Na₂SO₄, filtered and concentrated. The resulting residue waspurified via reverse phase chromatography (C18, CH₃CN:H₂O=6.5: 3.5) toafford(21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone(0.155 g, 28%) as white solid. ESI-MS (EI⁺, m/z): 1098.4 [M+Na]⁺. ¹H NMR(500 MHz, CDCl₃) δ 6.42-5.87 (m, 4H), 5.63-5.34 (m, 2H), 5.32-5.00 (m,2H), 4.85 (s, 1H), 4.35-4.09 (m, 1H), 4.05-3.49 (m, 18H), 3.49-3.01 (m,14H), 2.66 (dddd, J=31.3, 24.8, 21.2, 13.0 Hz, 4H), 2.33 (d, J=12.0 Hz,2H), 2.06 (dd, J=39.9, 10.6 Hz, 3H), 1.77-1.53 (m, 13H), 1.51-1.14 (m,10H), 1.14-0.81 (m, 18H), 0.71 (dd, J=23.8, 11.9 Hz, 1H).

Step 2: Synthesis of(21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,45S,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone(I-110) and(21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,45R,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone(I-109)

170 mg of(21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentonepurified via prep chiral HPLC and the resulting epimers purified viasilica gel chromatography (hexane:DCM:EtOAc:MeOH=3:3:1:0.8) to obtain(21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,45S,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone(I-110: 37 mg, 21% yield) and(21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,45R,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone(I-109: 33 mg, 19% yield), both as white solids.

Chiral Analysis Method:

Column: CHIRALPAK IC(IC00CE-OL002)

Column size: 0.46 cm I.D.×25 cm L

Injection: 30.0 ul

Mobile phase: Hexane/EtOH=60/40(V/V)

Flow rate: 1.0 ml/min

Wave length: UV 254 nm

Temperature: 35° C.

HPLC equipment: Shimadzu LC-20AT CP-HPLC-06

I-110: ESI-MS (EI⁺, m/z): 1098.0 [M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ6.41-6.18 (m, 2H), 6.12 (dd, J=15.0, 10.3 Hz, 1H), 5.93 (dd, J=33.7,10.6 Hz, 1H), 5.47 (ddd, J=33.0, 20.8, 9.5 Hz, 2H), 5.26 (d, J=5.6 Hz,1H), 5.13 (dt, J=21.8, 10.8 Hz, 1H), 4.88 (s, 1H), 4.19 (t, J=9.3 Hz,1H), 3.94-3.52 (m, 19H), 3.49-3.25 (m, 12H), 3.24-3.02 (m, 3H), 2.76(ddd, J=26.2, 16.6, 10.3 Hz, 3H), 2.57 (dd, J=17.0, 6.3 Hz, 1H), 2.29(t, J=26.2 Hz, 2H), 2.16-1.85 (m, 6H), 1.74-1.53 (m, 10H), 1.53-1.16 (m,9H), 1.15-1.01 (m, 8H), 1.01-0.82 (m, 10H), 0.71 (dd, J=23.9, 12.0 Hz,1H).

I-109: ESI-MS (EI⁺, m/z): 1098.0 [M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ6.43-5.90 (m, 4H), 5.62-5.02 (m, 5H), 4.24 (d, J=63.6 Hz, 1H), 3.97 (dd,J=21.5, 6.8 Hz, 1H), 3.86-3.50 (m, 18H), 3.45-3.01 (m, 14H), 2.73-2.46(m, 3H), 2.39-1.94 (m, 6H), 1.91-1.69 (m, 10H), 1.50-1.31 (m, 12H),1.16-0.85 (m, 18H), 0.69 (d, J=11.7 Hz, 1H).

Example 26: Synthesis of(30E,32E,34E,35E,38R,39S,40R,41R,43S,45S,47R,48S,50R,51R,60R)-50,60-dihydroxy-48-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-51-methoxy-38,39,40,41,52,53-hexamethyl-47-(1,4,7,10,13-pentaoxacyclopentadec-2-ylmethoxy)-75,76-dioxa-61-azatricyclohexatriaconta-30,32,34(52),35(53)-tetraene-54,55,56,57,58-pentone(I-107):(30E,32E,34E,35E,38R,39S,40R,41R,43S,45S,47S,48S,50R,51R,60R)-50,60-dihydroxy-48-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-51-methoxy-38,39,40,41,52,53-hexamethyl-47-(1,4,7,10,13-pentaoxacyclopentadec-2-ylmethoxy)-75,76-dioxa-61-azatricyclohexatriaconta-30,32,34(52),35(53)-tetraene-54,55,56,57,58-pentone(I-108)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of(30E,32E,34E,35E,38R,39S,40R,41R,43S,45S,48S,50R,51R,60R)-50,60-dihydroxy-48-[(1R)-2-[(1S, 3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-51-methoxy-38,39,40,41,52,53-hexamethyl-47-(1,4,7,10,13-pentaoxacyclopentadec-2-ylmethoxy)-75,76-dioxa-6l-azatricyclohexatriaconta-30,32,34(52),35(53)-tetraene-54,55,56,57,58pentone

A solution of rapamycin (2 g, 2.19 mmol),1,4,7,10,13-pentaoxacyclopentadec-2-ylmethanol (3.83 g, 15.31 mmol) andp-toluenesulfonic acid monohydrate (1.88 g, 10.94 mmol) in THF (10 mL)was stirred at 20° C. for 2 h. The mixture was then poured into ice coldsat.NaHCO₃(50 mL) and extracted with EtOAc (100 mL×3). The combinedorganic layers were washed with water and brine, then dried in vacuo andthe resulting residue purified via reverse phase chromatography (C18,CH₃CN:H₂O=8:2) to afford(30E,32E,34E,35E,38R,39S,40R,41R,43S,45S,48S,50R,51R,60R)-50,60-dihydroxy-48-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-51-methoxy-38,39,40,41,52,53-hexamethyl-47-(1,4,7,10,13-pentaoxacyclopentadec-2-ylmethoxy)-75,76-dioxa-61-azatricyclohexatriaconta-30,32,34(52),35(53)-tetraene-54,55,56,57,58-pentone(80 mg, 2.6% yield) as a white solid. ESI-MS (EI⁺, m/z): 1154.0 [M+Na]⁺.¹H NMR (500 MHz, CDCl₃) δ 6.16 (tdt, J=40.0, 33.8, 20.7 Hz, 4H),5.54-5.03 (m, 4H), 4.21 (t, J=22.5 Hz, 1H), 3.90-3.46 (m, 22H),3.44-3.04 (m, 12H), 2.74 (dddd, J=27.8, 22.2, 13.7, 4.7 Hz, 5H),2.37-1.56 (m, 22H), 1.50-1.16 (m, 8H), 1.13-0.81 (m, 18H), 0.67 (dd,J=23.8, 11.9 Hz, 1H).

Step 2: Synthesis of(30E,32E,34E,35E,38R,39S,40R,41R,43S,45S,47S,48S,50R,51R,60R)-50,60-dihydroxy-48-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-51-methoxy-38,39,40,41,52,53-hexamethyl-47-(1,4,7,10,13-pentaoxacyclopentadec-2-ylmethoxy)-75,76-dioxa-61-azatricyclohexatriaconta-30,32,34(52),35(53)-tetraene-54,55,56,57,58-pentone(I-108) and(30E,32E,34E,35E,38R,39S,40R,41R,43S,45S,47R,48S,50R,51R,60R)-50,60-dihydroxy-48-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-51-methoxy-38,39,40,41,52,53-hexamethyl-47-(1,4,7,10,13-pentaoxacyclopentadec-2-ylmethoxy)-75,76-dioxa-61-azatricyclohexatriaconta-30,32,34(52),35(53)-tetraene-54,55,56,57,58-pentone(I-107)

130 mg of(30E,32E,34E,35E,38R,39S,40R,41R,43S,45S,48S,50R,51R,60R)-50,60-dihydroxy-48-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-51-methoxy-38,39,40,41,52,53-hexamethyl-47-(1,4,7,10,13-pentaoxacyclopentadec-2-ylmethoxy)-75,76-dioxa-61-azatricyclohexatriaconta-30,32,34(52),35(53)-tetraene-54,55,56,57,58-pentonewas purified via prep chiral HPLC and the resulting epimers purified viasilica gel chromatography (hexane:DCM:EtOAc:MeOH=3:3:1:0.5) to obtain(30E,32E,34E,35E,38R,39S,40R,41R,43S,45S,47S,48S,50R,51R,60R)-50,60-dihydroxy-48-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-51-methoxy-38,39,40,41,52,53-hexamethyl-47-(1,4,7,10,13-pentaoxacyclopentadec-2-ylmethoxy)-75,76-dioxa-61-azatricyclohexatriaconta-30,32,34(52),35(53)-tetraene-54,55,56,57,58-pentone(I-108: 18 mg, 13% yield) and(30E,32E,34E,35E,38R,39S,40R,41R,43S,45S,47R,48S,50R,51R,60R)-50,60-dihydroxy-48-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-51-methoxy-38,39,40,41,52,53-hexamethyl-47-(1,4,7,10,13-pentaoxacyclopentadec-2-ylmethoxy)-75,76-dioxa-61-azatricyclohexatriaconta-30,32,34(52),35(53)-tetraene-54,55,56,57,58-pentone(I-107: 16 mg, 12% yield), both as white solids.

Chiral Analysis Method:

Column: CHIRALPAK IC(IC00CE-OL002)

Column size: 0.46 cm I.D.×25 cm L

Injection: 100.0 ul

Mobile phase: Hexane/EtOH=60/40(V/V)

Flow rate: 1.0 ml/min

Wave length: UV 254 nm

Temperature: 35° C.

HPLC equipment: Shimadzu LC-20AT CP-HPLC-06

I-108: ESI-MS (EI⁺, m/z): 1153.9 [M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ 6.31(dt, J=25.1, 14.5 Hz, 2H), 6.17-6.08 (m, 1H), 5.95 (s, 1H), 5.55-5.37(m, 2H), 5.26 (s, 1H), 5.16 (d, J=4.4 Hz, 1H), 4.71 (d, J=28.0 Hz, 1H),4.19 (s, 1H), 3.93-3.48 (m, 25H), 3.44-3.26 (m, 12H), 3.20-3.03 (m, 1H),2.94 (dd, J=16.5, 7.7 Hz, 1H), 2.70 (dd, J=17.9, 12.1 Hz, 3H), 2.56 (d,J=17.0 Hz, 1H), 2.33 (d, J=12.0 Hz, 2H), 2.13-1.84 (m, 5H), 1.81-1.67(m, 8H), 1.50-1.17 (m, 8H), 1.14-0.81 (m, 20H), 0.67 (dd, J=23.6, 11.9Hz, 1H).

I-107: ESI-MS (EI⁺, m/z): 1153.9 [M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ6.40-5.95 (m, 4H), 5.58-5.11 (m, 4H), 4.30-4.12 (m, 1H), 3.88-3.51 (m,25H), 3.42-3.12 (m, 12H), 2.99-2.53 (m, 5H), 2.50-1.90 (m, 5H),1.83-1.65 (m, 14H), 1.44-1.30 (m, 8H), 1.12-0.76 (m, 18H), 0.74-0.62 (m,1H).

Example 27: Synthesis of(21E,23E,25E,26E,31R,32S,33R,34R,36S,38S,41S,42R,43R,52R)-40-[bis(hydroxymethyl)phosphorylmethoxy]-42,52-dihydroxy-41-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-43-methoxy-31,32,33,34,44,45-hexamethyl-65,66-dioxa-53-azatricyclohexatriaconta-21,23,25(44),26(45)-tetraene-46,47,48,49,50-pentone(I-106)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis ofBis(hydroxymethyl)phosphorylmethanol

Ba(OH)₂ (8.43 g, 49.23 mmol) was dissolved in 50 mL of distilled waterat 60° C. Tetrakis(hydroxymethyl)phosphonium sulfate (20 g, 49.23 mmol)was added dropwise to the solution which was then stirred at 60° C. for4 h. After the BaSO₄ was removed via centrifugation, hydrogen peroxide(30% solution, 98.45 mmol) was added slowly and the resulting reactionstirred at room temperature for 5 h. The mixture was washed withchloroform then the desired product obtained by removing the water underreduced pressure (6.5 g, 94% yield) as an oil. ESI-MS (EI⁺, m/z): 141.1[M+H]⁺. ¹HNMR (500 MHz, DMSO-d₆): δ 5.35 (bs, 3H), 3.97 (s, 6H).

Step 2: Synthesis of(21E,23E,25E,26E,31R,32S,33R,34R,36S,38S,41S,42R,43R,52R)-40-[bis(hydroxymethyl)phosphorylmethoxy]-42,52-dihydroxy-41-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-43-methoxy-31,32,33,34,44,45-hexamethyl-65,66-dioxa-53-azatricyclohexatriaconta-21,23,25(44),26(45)-tetraene-46,47,48,49,50-pentone(I-106)

A mixture of rapamycin (0.5 g, 0.547 mmol),bis(hydroxymethyl)phosphorylmethanol (0.766 g, 5.47 mmol) and4-methylbenzenesulfonic acid hydrate (0.52 g, 2.73 mmol) in THF (20 mL)was stirred at 25° C. for 4h. EtOAc (100 mL) and water (50 mL) wereadded. The aqueous layer was extracted with EtOAc (100 mL×2) and thecombined organic layers washed with water (2×50 mL) and brine (50 mL)then dried over anhydrous Na₂SO₄, filtered and concentrated. Theresulting residue was purified via reverse phase chromatography (C18,CH₃CN:H₂O=7:3) to afford(21E,23E,25E,26E,31R,32S,33R,34R,36S,38S,41S,42R,43R,52R)-40-[bis(hydroxymethyl)phosphorylmethoxy]-42,52-dihydroxy-41-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-43-methoxy-31,32,33,34,44,45-hexamethyl-65,66-dioxa-53-azatricyclohexatriaconta-21,23,25(44),26(45)-tetraene-46,47,48,49,50-pentone(I-106: 50 mg, 9% yield) as a white solid. ESI-MS (EI⁺, m/z): 1043.9[M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ 6.47-5.91 (m, 4H), 5.58-5.08 (m, 4H),4.51-3.63 (m, 12H), 3.59-3.16 (m, 14H), 2.80 (d, J=138.1 Hz, 6H),2.38-1.91 (m, 8H), 1.50-0.77 (m, 35H), 0.67 (d, J=9.0 Hz, 1H).

Example 28: Synthesis of(22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,44R,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-44-[2-(2-methoxyethoxy)ethoxy]-35,36,37,38,48,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone(I-104) and(22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,44S,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-44-[2-(2-methoxyethoxy)ethoxy]-35,36,37,38,48,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone(I-105)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of(22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-44-[2-(2-methoxyethoxy)ethoxy]-35,36,37,38,48,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone

A mixture of everolimus (5 g, 5.22 mmol) and 2-(2-methoxyethoxy)ethanol(15 mL) in THF (80 mL) was degassed with N₂ then heated at 50° C. HND-8(600 mg) was added and the resulting mixture stirred at 50° C. for 4 hunder N₂ then filtered and diluted with EtOAc. After concentration, theresidue was purified by reverse phase chromatography (C18, CH₃CN: H₂Ofrom 0%-100% yield) to afford(22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-44-[2-(2-methoxyethoxy)ethoxy]-35,36,37,38,48,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone(1.5 g, 27% yield) as a white solid. ESI-MS (EI⁺, m/z): 1068.4 [M+Na]⁺.¹H NMR (400 MHz, CDCl₃) δ 6.40-5.85 (m, 4H), 5.56-5.36 (m, 2H), 5.21(ddd, J=16.0, 11.7, 5.6 Hz, 2H), 4.21 (dd, J=25.8, 15.5 Hz, 2H),3.94-3.26 (m, 28H), 3.25-3.02 (m, 4H), 2.76-2.40 (m, 3H), 2.34 (d,J=13.3 Hz, 2H), 2.19-2.06 (m, 2H), 2.01-1.67 (m, 13H), 1.54-1.30 (m,7H), 1.15-0.81 (m, 18H), 0.72 (dd, J=23.1, 11.7 Hz, 1H).

Step 2: Synthesis of(22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,44S,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-44-[2-(2-methoxyethoxy)ethoxy]-35,36,37,38,48,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone(I-105) and(22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,44R,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-44-[2-(2-methoxyethoxy)ethoxy]-35,36,37,38,48,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone(I-104)

120 mg of(22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-44-[2-(2-methoxyethoxy)ethoxy]-35,36,37,38,48,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentonewas purified via prep chiral HPLC to obtain(22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,44S,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-44-[2-(2-methoxyethoxy)ethoxy]-35,36,37,38,48,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone(I-105: 17 mg, 20% yield) and(22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,44R,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-44-[2-(2-methoxyethoxy)ethoxy]-35,36,37,38,48,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone(I-104: 15 mg, 16% yield), both as white solids.

Chiral Separation Method:

Column: CHIRALPAK IC

Column size: 2.5 cm I.D.×25 cm L, 10 μm

Sample solution: 14 mg/ml in Mobile phase

Injection: 15 ml

Mobile phase: Hexane/EtOH=50/50(V/V)

Flow rate: 60 ml/min

Wave length: UV 254 nm

Temperature: 35° C.

I-105: ESI-MS (EI⁺, m/z): 1068.4 [M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ6.42-6.19 (m, 2H), 6.13 (dd, J=15.1, 10.0 Hz, 1H), 5.91 (dd, J=33.0,10.5 Hz, 1H), 5.56-5.38 (m, 2H), 5.27 (d, J=5.0 Hz, 1H), 5.15 (dt,J=15.2, 7.6 Hz, 1H), 4.76 (s, 1H), 4.18 (d, J=5.6 Hz, 1H), 3.93-3.25 (m,30H), 3.24-3.03 (m, 3H), 2.72 (dd, J=16.7, 5.6 Hz, 2H), 2.57 (dd,J=16.8, 6.5 Hz, 1H), 2.34 (d, J=13.9 Hz, 2H), 2.13-1.84 (m, 6H),1.82-1.67 (m, 7H), 1.47 (dd, J=24.1, 16.7 Hz, 4H), 1.25 (ddd, J=24.1,20.2, 10.0 Hz, 7H), 1.14-0.81 (m, 18H), 0.72 (dd, J=23.9, 12.1 Hz, 1H).

I-104: ESI-MS (EI⁺, m/z): 1068.4 [M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ 6.19(m, 4H), 5.56-5.36 (m, 2H), 5.28-5.07 (m, 2H), 4.83 (d, J=4.9 Hz, 4H),4.28 (s, 1H), 4.21-4.09 (m, 1H), 4.04-3.51 (m, 15H), 3.46-3.29 (m, 11H),3.27-2.91 (m, 5H), 2.76-2.42 (m, 3H), 2.31 (d, J=11.3 Hz, 2H), 2.18-1.70(m, 13H), 1.53-1.19 (m, 8H), 1.16-0.84 (m, 18H), 0.76-0.60 (m, 1H).

Example 29: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethylsulfonyl)ethylsulfonyl]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-95) and(21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethylsulfonyl)ethylsulfonyl]ethoxy]-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-102)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethylsulfanyl)ethylsulfanyl]ethoxy]-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone

To a mixture of rapamycin (1 g, 1.09 mmol),2-[2-(2-hydroxyethylsulfanyl)ethylsulfanyl]ethanol (2 g, 10.94 mmol) inTHF (20 mL) was added 4-methylbenzenesulfonic acid monohydrate (0.62 g,3.28 mmol) at 15° C. The resulting mixture was stirred at 15° C. for 17h then diluted with EtOAc (100 mL) and adjusted to pH 9 using saturatedaqueous NaHCO₃ solution (about 50 mL). The organic layer was thenconcentrated and the residue purified by reverse phase chromatography(C18, CH₃CN:H₂O=6:4) to provide(21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethylsulfanyl)ethylsulfanyl]ethoxy]-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(150 mg, 12% yield) as a yellow solid. ESI-MS (EI⁺, m/z): 1086.4[M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ 6.39-5.95 (m, 4H), 5.54-5.19 (m, 4H),4.81-4.17 (m, 2H), 3.96-3.73 (m, 4H), 3.59-3.14 (m, 12H), 2.96-2.55 (m,14H), 2.35-1.87 (m, 6H), 1.81-1.59 (m, 13H), 1.53-1.13 (m, 11H),1.16-0.84 (m, 18H), 0.71-0.63 (m, 1H).

Step 2: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethylsulfonyl)ethylsulfonyl]ethoxy]-44-[(S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-102)

To a solution of(21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethylsulfanyl)ethylsulfanyl]ethoxy]-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(170 mg, 0.16 mmol) in methanol (8 mL) was added Oxone (393 mg, 0.64mmol) at 0° C. The resulting mixture was allowed to warm to 10° C. andstirred for 5 h. The reaction was filtered then purified via reversephase chromatography (C18, using a 5-60% acetonitrile-water) to afford(21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethylsulfonyl)ethylsulfonyl]ethoxy]-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-102, 30 mg, 17% yield) as a white solid. ESI-MS (EI⁺, m/z): 1150.8[M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ 6.40-5.90 (m, 4H), 5.57-5.08 (m, 5H),4.14 (s, 4H), 3.68 (tdd, J=37.6, 33.2, 11.6 Hz, 11H), 3.48-3.13 (m,20H), 2.95 (s, 2H), 2.68 (dd, J=36.4, 30.5 Hz, 5H), 2.37-1.70 (m, 12H),1.31 (dd, J=78.6, 46.8 Hz, 7H), 1.13-0.81 (m, 18H), 0.67 (d, J=11.9 Hz,1H).

Step 3: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethylsulfonyl)ethylsulfonyl]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-95)

90 mg of(21E,23E,25E,26E,34R,35S,36R,37R,39R,41S,44S,45R,46R,55S)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethylsulfonyl)ethylsulfonyl]ethoxy]-44-[(1S)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentonewas purified via prep chiral HPLC to provide(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethylsulfonyl)ethylsulfonyl]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-95: 15 mg, 16% yield) as a white solid.

Chiral Analysis Method:

Column: CHIRALPAK IC(IC00CD-NA012)

Column size: 0.46 cm I.D.×15 cm L

Injection: 10.0 ul

Mobile phase: EtOH=100%

Flow rate: 0.5 ml/min

Wave length: UV 254 nm

Temperature: 35° C.

HPLC equipment: Shimadzu LC-20AD CP-HPLC-06

I-95: ESI-MS (EI⁺, m/z): 1150.3 [M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ 6.32(td, J=24.8, 14.8 Hz, 2H), 6.13 (dd, J=14.9, 9.9 Hz, 1H), 5.98 (dd,J=22.1, 10.3 Hz, 1H), 5.56-5.31 (m, 2H), 5.26 (d, J=5.4 Hz, 1H), 5.14(d, J=4.1 Hz, 1H), 4.86 (s, 1H), 4.16 (dd, J=11.9, 5.5 Hz, 3H),3.92-3.49 (m, 11H), 3.44-3.17 (m, 15H), 2.93 (dd, J=14.1, 5.5 Hz, 1H),2.78-2.50 (m, 5H), 2.36-2.17 (m, 2H), 2.01 (ddd, J=21.5, 18.0, 9.0 Hz,5H), 1.84-1.65 (m, 11H), 1.49-1.16 (m, 12H), 1.14-0.82 (m, 14H), 0.66(dd, J=23.8, 12.0 Hz, 1H).

Example 29: Synthesis of3-[2,2-bis(2-cyanoethoxymethyl)-3-[[(21E,23E,25E,26E,41R,42S,43R,44R,46S,48S,51S,52R,53R,62R)-52,62-dihydroxy-51-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-53-methoxy-41,42,43,44,54,55-hexamethyl-56,57,58,59,60-pentaoxo-76,77-dioxa-67-azatricyclohexatriaconta-21,23,25(54),26(55)-tetraen-50-yl]oxy]propoxy]propanenitrile(I-101)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of(1-methyl-2,6,7-trioxabicyclo[2.2.2]octan-4-yl)methanol

To a solution of 2,2-bis(hydroxymethyl)propane-1,3-diol (20 g, 146.9mmol) and 4-methylbenzenesulfonic acid (0.25 g, 1.47 mmol) in toluene(200 mL) was added 1,1,1-triethoxyethane (27 mL, 146.9 mmol) at reflux.The reaction was then stirred at 130° C. until the solution becameclear, a few drops of TEA were added and the reaction filtered whilestill hot. The filtrate was cooled then concentrated to provide(1-methyl-2,6,7-trioxabicyclo[2.2.2]octan-4-yl)methanol (20 g, 85%yield) as colorless crystals. ¹HNMR (500 MHz, DMSO-d₆): δ 4.79 (t, J=5.3Hz, 1H), 3.85 (s, 6H), 3.22 (d, J=5.3 Hz, 2H), 1.29 (s, 3H).

Step 2: Synthesis of4-(benzyloxymethyl)-1-methyl-2,6,7-trioxabicyclo[2.2.2]octane

Solid (1-methyl-2,6,7-trioxabicyclo[2.2.2]octan-4-yl)methanol (1.5 g,9.37 mmol) was added to a stirred suspension of finely powered KOH (2.47g, 44 mmol) and BnBr (1.86 g, 10.86 mmol) in DMSO (5 mL). The resultingsolution was stirred at rt for 1h then poured into ice water andextracted with EtOAc, dried, filtered and concentrated. The residue waspurified via silical gel chromatography (EtOAc:PE=1:10) to afford4-(benzyloxymethyl)-1-methyl-2,6,7-trioxabicyclo[2.2.2]octane (1.5 g,64% yield) as a light yellow solid. ESI-MS (EI⁺, m/z): 251.1 [M+H]⁺. ¹HNMR (500 MHz, CDCl₃) δ 7.36-7.25 (m, 5H), 4.45 (s, 2H), 4.01 (s, 6H),3.19 (s, 2H), 1.45 (s, 3H).

Step 3: Synthesis of2-(benzyloxymethyl)-2-(hydroxymethyl)propane-1,3-diol

A solution of4-(benzyloxymethyl)-1-methyl-2,6,7-trioxabicyclo[2.2.2]octane (2 g, 8mmol) and hydrogen chloride (2 mL, 2M in water) in THF (20 mL) wasstirred at rt overnight then concentrated and purified by reverse phasechromatography (C18, CH₃CN:H₂O=1:3) to provide2-(benzyloxymethyl)-2-(hydroxymethyl)propane-1,3-diol (1.3 g, 72% yield)as a thick yellow oil. ESI-MS (EI⁺, m/z): 227.1 [M+H]⁺. ¹H NMR (500 MHz,CDCl₃) δ 7.36-7.26 (m, 5H), 4.48 (s, 2H), 3.68 (s, 6H), 3.46 (s, 2H),3.40 (bs, 3H).

Step 4: Synthesis of3-[2-(benzyloxymethyl)-3-(2-cyanoethoxy)-2-(2-cyanoethoxymethyl)propoxy]propanenitrile

To a mixture of 2-(benzyloxymethyl)-2-(hydroxymethyl)propane-1,3-diol(4.9 g, 21.66 mmol) and KOH (98 mg, 1.75 mmol) was slowly addedacrylonitrile (11.49 g, 216.56 mmol), ensuring that the internalreaction temperature did not exceed 30° C. The mixture was then stirredovernight at room temperature, neutralized with 1 N HCl aqueous solutionand extracted with EtOAc (200 mL). The combined organic layers werewashed twice with water, dried over anhydrous Na₂SO₄, filtered andconcentrated to obtain3-[2-(benzyloxymethyl)-3-(2-cyanoethoxy)-2-(2-cyanoethoxymethyl)propoxy]propanenitrile(7 g, 84% yield) as a yellow solid. ESI-MS (EI⁺, m/z): 386.3 [M+H]⁺.

Step 5: Synthesis of3-[2,2-bis(2-cyanoethoxymethyl)-3-hydroxy-propoxy]propanenitrile

To a solution of3-[2-(benzyloxymethyl)-3-(2-cyanoethoxy)-2-(2-cyanoethoxymethyl)propoxy]propanenitrile(5 g, 12.97 mmol) in MeOH (50 mL) was added Pd/C (1.59 g). The mixturewas stirred at room temperature overnight under a balloon of hydrogenthen filtered through a pad of celite and washed with ethanol. Theresulting solution was concentrated to provide3-[2,2-bis(2-cyanoethoxymethyl)-3-hydroxy-propoxy]propanenitrile (2.6 g,68% yield) as a colorless oil. ESI-MS (EI⁺, m/z): 296.2 [M+H]⁺.

Step 6: Synthesis of3-[2,2-bis(2-cyanoethoxymethyl)-3-[[(21E,23E,25E,26E,41R,42S,43R,44R,46S,48S,51S,52R,53R,62R)-52,62-dihydroxy-51-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-53-methoxy-41,42,43,44,54,55-hexamethyl-56,57,58,59,60-pentaoxo-76,77-dioxa-67-azatricyclohexatriaconta-21,23,25(54),26(55)-tetraen-50-yl]oxy]propoxy]propanenitrile(I-101)

To a solution of rapamycin (0.5 g, 0.547 mmol) in DCM (30 mL) was added2,2,2-trifluoroacetic acid (2.4 mL) at −40° C. The reaction was stirredat −40° C. for 10 min, then3-[2,2-bis(2-cyanoethoxymethyl)-3-hydroxy-propoxy]propanenitrile (0.48g, 1.64 mmol) was added. After stirring for a further 1 h, the reactionwas diluted with DCM (3 ml) and poured into a cold NaHCO₃ aqueoussolution. The organic layer was washed with water and brine, dried overNa₂SO₄, filtered and concentrated. The residue was purified by reversephase chromatography (C18, CH₃CN:H₂O=8:2) to provide3-[2,2-bis(2-cyanoethoxymethyl)-3-[[(21E,23E,25E,26E,41R,42S,43R,44R,46S,48S,51S,52R,53R,62R)-52,62-dihydroxy-51-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-53-methoxy-41,42,43,44,54,55-hexamethyl-56,57,58,59,60-pentaoxo-76,77-dioxa-67-azatricyclohexatriaconta-21,23,25(54),26(55)-tetraen-50-yl]oxy]propoxy]propanenitrile(I-101: 80 mg, 12% yield) as a white solid. ESI-MS (EI⁺, m/z): 1198.8[M+Na]⁺. ¹H NMR (500 MHz, CDCl3) δ 6.42-5.87 (m, 4H), 5.35 (ddd,J=120.9, 41.2, 32.5 Hz, 4H), 4.24 (dd, J=29.5, 16.4 Hz, 2H), 3.98 (d,J=4.2 Hz, 1H), 3.87-3.60 (m, 7H), 3.56-3.01 (m, 18H), 2.88 (d, J=59.1Hz, 2H), 2.74-2.42 (m, 9H), 2.34 (s, 2H), 2.23-1.84 (m, 5H), 1.82-1.65(m, 13H), 1.53-1.22 (m, 10H), 1.16-0.84 (m, 18H), 0.72-0.61 (m, 1H).

Example 30: Synthesis of(21E,23E,25E,26E,37R,38S,39R,40R,42S,44S,47S,48R,49R,59R)-48,59-dihydroxy-46-[2-[3-(2-hydroxyethoxy)-2-(2-hydroxyethoxymethyl)propoxy]ethoxy]-47-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-49-methoxy-37,38,39,40,50,51-hexamethyl-71,72-dioxa-60-azatricyclohexatriaconta-21,23,25(50),26(51)-tetraene-52,53,54,55,56-pentone(I-100) and(21E,23E,25E,26E,37R,38S,39R,40R,42S,44S,46R,47S,48R,49R,59R)-48,59-dihydroxy-46-[2-[3-(2-hydroxyethoxy)-2-(2-hydroxyethoxymethyl)propoxy]ethoxy]-47-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-49-methoxy-37,38,39,40,50,51-hexamethyl-71,72-dioxa-60-azatricyclohexatriaconta-21,23,25(50),26(51)-tetraene-52,53,54,55,56-pentone(I-64) and(21E,23E,25E,26E,37R,38S,39R,40R,42S,44S,46S,47S,48R,49R,59R)-48,59-dihydroxy-46-[2-[3-(2-hydroxyethoxy)-2-(2-hydroxyethoxymethyl)propoxy]ethoxy]-47-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]l-1-methyl-ethyl]-49-methoxy-37,38,39,40,50,51-hexamethyl-71,72-dioxa-60-azatricyclohexatriaconta-21,23,25(50),26(51)-tetraene-52,53,54,55,56-pentone(I-65)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of7-((2-(benzyloxy)ethoxy)methyl)-1,13-diphenyl-2,5,9,12-tetraoxatridecane

To a solution of 2-(hydroxymethyl)propane-1,3-diol (2 g, 18.85 mmol) inTHF (30 mL) at 0° C. was added sodium hydride (9.05 g, 376.93 mmol). andthe reaction heated to 50° C. for 1 h then cooled to rt.2-bromoethoxymethylbenzene (40.54 g, 188.47 mmol) was then added and themixture heated to 65° C. for 17 h. The reaction was quenched withice-water (50 mL), then this was extracted with EtOAc (50 mL×2). Thecombined organic layers were washed with brine (100 mL), dried,filtered, concentrated and then purified by silica gel chromatography(PE:EtOAc=20:1) to afford2-[3-(2-benzyloxyethoxy)-2-(2-benzyloxyethoxymethyl)propoxy]ethoxymethylbenzene(5 g, 52% yield) as a colorless liquid. ESI-MS (EI⁺, m/z): 509.0 [M+H]⁺.

Step 2: Synthesis of2,2′-(2-((2-hydroxyethoxy)methyl)propane-,3-diyl)bis(oxy)diethanol

To a solution of2-[3-(2-benzyloxyethoxy)-2-(2-benzyloxyethoxymethyl)propoxy]ethoxymethylbenzene(2 g, 3.93 mmol) in MeOH (20 mL) was added Pd/C (2.41 g). The mixturewas stirred at room temperature overnight under a balloon of hydrogen.The reaction mixture was then filtered through a pad of celite and whichwas then washed with ethanol. The resulting solution was concentratedunder reduced pressure to provide2,2′-((2-((2-hydroxyethoxy)methyl)propane-1,3-diyl)bis(oxy))bis(ethan-1-ol)(0.92 g, 98% yield). ¹HNMR (500 MHz, CDCl₃) δ 3.65 (dd, J=5.7, 3.4 Hz,6H), 3.53-3.48 (m, 12H), 2.22-2.14 (m, 1H).

Step 3: Synthesis of(21E,23E,25E,26E,37R,38S,39R,40R,42S,44S,47S,48R,49R,59R)-48,59-dihydroxy-46-[2-[3-(2-hydroxyethoxy)-2-(2-hydroxyethoxymethyl)propoxy]ethoxy]-47-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-49-methoxy-37,38,39,40,50,51-hexamethyl-71,72-dioxa-60-azatricyclohexatriaconta-21,23,25(50),26(51)-tetraene-52,53,54,55,56-pentone(I-100)

To a solution of rapamycin (0.5 g, 0.547 mmol) and4-methylbenzenesulfonic acid (0.47 g, 2.73 mmol) in THF (10 mL) wasadded 2-[3-(2-hydroxyethoxy)-2-(2-hydroxyethoxymethyl)propoxy]ethanol(1.3 g, 5.47 mmol). The mixture was stirred at 25° C. for 2 h thenpoured into ice-cold NaHCO₃ aqueous solution and extracted with EtOAc.The organic layer was dried, filtered and concentrated. The residue waspurified by reverse phase chromatography (C18, CH₃CN: H₂O: 7:3) followedby silica gel chromatography (DCM:MeOH=15:1) to provide(21E,23E,25E,26E,37R,38S,39R,40R,42S,44S,47S,48R,49R,59R)-48,59-dihydroxy-46-[2-[3-(2-hydroxyethoxy)-2-(2-hydroxyethoxymethyl)propoxy]ethoxy]-47-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-49-methoxy-37,38,39,40,50,51-hexamethyl-71,72-dioxa-60-azatricyclohexatriaconta-21,23,25(50),26(51)-tetraene-52,53,54,55,56-pentone(I-100, 150 mg, 25% yield) as a white solid. ESI-MS (EI⁺, m/z): 1142.0[M+Na]⁺. ¹H NMR (500 MHz, CDCl3) δ 6.21 (dddd, J=32.0, 27.8, 21.3, 10.2Hz, 3H), 6.05-5.84 (m, 1H), 5.57-5.36 (m, 2H), 5.29-4.97 (m, 2H), 4.83(s, 1H), 4.20 (dd, J=36.3, 30.2 Hz, 1H), 4.01-3.66 (m, 6H), 3.62-3.22(m, 29H), 3.00-2.43 (m, 9H), 2.36-1.85 (m, 9H), 1.77-1.51 (m, 6H),1.52-1.17 (m, 9H), 1.16-0.79 (m, 18H), 0.65 (dt, J=21.9, 11.0 Hz, 1H).

Step 4: Synthesis of(21E,23E,25E,26E,37R,38S,39R,40R,42S,44S,46S,47S,48R,49R,59R)-48,59-dihydroxy-46-[2-[3-(2-hydroxyethoxy)-2-(2-hydroxyethoxymethyl)propoxy]ethoxy]-47-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-49-methoxy-37,38,39,40,50,51-hexamethyl-71,72-dioxa-60-azatricyclohexatriaconta-21,23,25(50),26(51)-tetraene-52,53,54,55,56-pentone(I-65) and(21E,23E,25E,26E,37R,38S,39R,40R,42S,44S,46R,47S,48R,49R,59R)-48,59-dihydroxy-46-[2-[3-(2-hydroxyethoxy)-2-(2-hydroxyethoxymethyl)propoxy]ethoxy]-47-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl-]1-methyl-ethyl]-49-methoxy-37,38,39,40,50,51-hexamethyl-71,72-dioxa-60-azatricyclohexatriaconta-21,23,25(50),26(51)-tetraene-52,53,54,55,56-pentone(I-64)

140 mg of(21E,23E,25E,26E,37R,38S,39R,40R,42S,44S,47S,48R,49R,59R)-48,59-dihydroxy-46-[2-[3-(2-hydroxyethoxy)-2-(2-hydroxyethoxymethyl)propoxy]ethoxy]-47-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-49-methoxy-37,38,39,40,50,51-hexamethyl-71,72-dioxa-60-azatricyclohexatriaconta-21,23,25(50),26(51)-tetraene-52,53,54,55,56-pentonewas purified via prep chiral HPLC and the resulting epimers purified viasilica gel chromatography (hexane:DCM:EtOAc:MeOH=3:3:1:1) to obtain(21E,23E,25E,26E,37R,38S,39R,40R,42S,44S,46S,47S,48R,49R,59R)-48,59-dihydroxy-46-[2-[3-(2-hydroxyethoxy)-2-(2-hydroxyethoxymethyl)propoxy]ethoxy]-47-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-49-methoxy-37,38,39,40,50,51-hexamethyl-71,72-dioxa-60-azatricyclohexatriaconta-21,23,25(50),26(51)-tetraene-52,53,54,55,56-pentone(I-65: 35.2 mg, 25% yield) and(21E,23E,25E,26E,37R,38S,39R,40R,42S,44S,46R,47S,48R,49R,59R)-48,59-dihydroxy-46-[2-[3-(2-hydroxyethoxy)-2-(2-hydroxyethoxymethyl)propoxy]ethoxy]-47-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-49-methoxy-37,38,39,40,50,51-hexamethyl-71,72-dioxa-60-azatricyclohexatriaconta-21,23,25(50),26(51)-tetraene-52,53,54,55,56-pentone(I-64:16 mg, 11% yield), both as white solids.

Chiral Separation Method:

Column: CHIRALPAK IC

Column size: 2.5 cm I.D.×25 cm L

Solution concentration: 1.4 mg/ml

Injection: 7 ml

Mobile phase: Hexane/EtOH=60/40(V/V)

Flow rate: 30 ml/min

Wave length: UV 254 nm

Temperature: 35° C.

I-65: ESI-MS (EI⁺, m/z): 1142.4 [M+Na]⁺. ¹H NMR (400 MHz, CDCl3) δ6.42-6.18 (m, 2H), 6.13 (dd, J=14.9, 10.1 Hz, 1H), 5.93 (dd, J=23.9,11.0 Hz, 1H), 5.54-4.89 (m, 6H), 4.80 (s, 1H), 4.18 (d, J=6.0 Hz, 1H),3.90 (s, 1H), 3.80-3.66 (m, 5H), 3.63-3.48 (m, 11H), 3.47-3.21 (m, 11H),2.99-2.47 (m, 6H), 2.38-2.15 (m, 3H), 2.14-1.85 (m, 5H), 1.84-1.64 (m,12H), 1.46 (dd, J=28.7, 18.7 Hz, 5H), 1.35-1.15 (m, 7H), 1.15-0.81 (m,18H), 0.66 (dd, J=23.7, 11.8 Hz, 1H).

I-64: ESI-MS (EI⁺, m/z): 1142.4 [M+Na]⁺. ¹H NMR (400 MHz, CDCl3) δ6.34-5.86 (m, 4H), 5.41 (ddd, J=54.0, 26.4, 18.2 Hz, 2H), 5.16 (dd,J=9.6, 5.0 Hz, 2H), 4.88 (s, 1H), 4.20 (d, J=11.0 Hz, 1H), 4.14-4.02 (m,1H), 3.93 (d, J=3.9 Hz, 1H), 3.87-3.72 (m, 1H), 3.64 (d, J=4.4 Hz, 4H),3.57-3.36 (m, 14H), 3.36-3.22 (m, 8H), 3.21-3.11 (m, 1H), 2.92-2.33 (m,8H), 2.32-2.11 (m, 3H), 2.11-1.85 (m, 5H), 1.82-1.63 (m, 11H), 1.48-1.25(m, 9H), 1.09-0.72 (m, 18H), 0.66-0.50 (m, 1H).

Example 31: Synthesis of(21E,23E,25E,26E,48R,49S,50R,51R,53S,55S,58S,59R,60R,69R)-59,69-dihydroxy-57-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-58-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-60-methoxy-48,49,50,51,61,62-hexamethyl-80,81-dioxa-70-azatricyclohexatriaconta-21,23,25(61),26(62)-tetraene-63,64,65,66,67-pentone(I-98)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of(21E,23E,25E,26E,48R,49S,50R,51R,53S,55S,58S,59R,60R,69R)-59,69-dihydroxy-57-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-58-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-60-methoxy-48,49,50,51,61,62-hexamethyl-80,81-dioxa-70-azatricyclohexatriaconta-21,23,25(61),26(62)-tetraene-63,64,65,66,67-pentone(I-98)

To a solution of rapamycin (0.5 g, 0.547 mmol) and4-methylbenzenesulfonic acid (0.47 g, 2.73 mmol) in THF (15 mL) wasadded2-[2-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol(2.51 g, 5.47 mmol) and the mixture stirred at 25° C. for 2 h. Thereaction was then poured into a cold aqueous NaHCO₃ solution which wasthen extracted with EtOAc. The organic layer was then dried over Na₂SO₄,filtered and concentrated. The residue was purified by reverse phasechromatography (CH₃CN:pure water=7:3) to provide(21E,23E,25E,26E,48R,49S,50R,51R,53S,55S,58S,59R,60R,69R)-59,69-dihydroxy-57-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-58-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-60-methoxy-48,49,50,51,61,62-hexamethyl-80,81-dioxa-70-azatricyclohexatriaconta-21,23,25(61),26(62)-tetraene-63,64,65,66,67-pentone(I-98: 115 mg, 15% yield) as a thick oil. ESI-MS (EI⁺, m/z): 1362.9[M+Na]⁺. ¹H NMR (500 MHz, CDCl3) δ 6.40-5.83 (m, 4H), 5.55-5.35 (m, 2H),5.32-5.03 (m, 2H), 4.31-4.10 (m, 1H), 3.93 (dd, J=70.7, 6.3 Hz, 1H),3.78-3.15 (m, 53H), 2.97-2.41 (m, 5H), 2.32 (s, 2H), 2.15-1.55 (m, 18H),1.52-1.16 (m, 10H), 1.14-0.81 (m, 18H), 0.73-0.58 (m, 1H).

Example 32: Synthesis of2-[[(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,43S,44R,45R,55R)-44,55-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-48,49,50,51,52-pentaoxo-67,68-dioxa-57-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraen-42-yl]oxy]ethylN-methylcarbamate (I-81) and2-[[(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,42R,43S,44R,45R,55R)-44,55-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-48,49,50,51,52-pentaoxo-67,68-dioxa-57-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraen-42-yl]oxy]ethylN-methylcarbamate (I-74) and2-[[(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,42S,43S,44R,45R,55R)-44,55-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-48,49,50,51,52-pentaoxo-67,68-dioxa-57-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraen-42-yl]oxy]ethylN-methylcarbamate (I-75)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of2-[[(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,43S,44R,45R,55R)-44,55-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-48,49,50,51,52-pentaoxo-67,68-dioxa-57-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraen-42-yl]oxy]ethylN-methylcarbamate (I-81)

To a degassed solution of everolimus (0.5 g, 0.52 mmol) in THF (10 mL)at 0° C., was added p-toluenesulfonic acid (0.45 g, 2.61 mmol) and2-hydroxyethyl N-methylcarbamate (2.80 mL). The resulting mixture wasstirred at 0° C. for 0.5 h under N₂, then warmed 23° C. and stirred for3h. The mixture was poured into sat.NaHCO₃(40 mL) which was extractedwith EtOAc (30 mL). The organic layer was washed with water (30 mL×2),brine (40 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated.The residue was purified by silica gel chromatography (DCM:MeOH=10:1)then additionally purified via reverse phase chromatography (C18,CH₃CN:H₂O=7:3) to provide2-[[(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,43S,44R,45R,55R)-44,55-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-48,49,50,51,52-pentaoxo-67,68-dioxa-57-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraen-42-yl]oxy]ethylN-methylcarbamate(I-81:100 mg, 18% yield) as a white solid. ESI-MS (EI⁺, m/z): 1067.4 [M+Na]⁺.¹H NMR (400 MHz, CDCl3) δ 6.50-5.91 (m, 4H), 5.58-4.97 (m, 4H), 4.70 (s,1H), 4.51 (d, J=40.4 Hz, 1H), 4.33-4.02 (m, 3H), 3.93-3.62 (m, 6H),3.61-3.00 (m, 13H), 2.86-2.46 (m, 6H), 2.40-2.22 (m, 2H), 2.18-1.69 (m,22H), 1.58-1.25 (m, 7H), 1.24-0.79 (m, 18H), 0.79-0.62 (m, 1H).

Step 2: Synthesis of2-[[(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,42S,43S,44R,45R,55R)-44,55-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-48,49,50,51,52-pentaoxo-67,68-dioxa-57-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraen-42-yl]oxy]ethylN-methylcarbamate (I-75) and2-[[(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,42R,43S,44R,45R,55R)-44,55-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-48,49,50,51,52-pentaoxo-67,68-dioxa-57-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraen-42-yl]oxy]ethylN-methylcarbamate (I-74)

120 mg of2-[[(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,43S,44R,45R,55R)-44,55-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-48,49,50,51,52-pentaoxo-67,68-dioxa-57-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraen-42-yl]oxy]ethylN-methylcarbamate was purified via prep chiral HPLC and the resultingepimers purified via silica gel chromatography(hexane:DCM:EtOAc:MeOH=3:3:1:0.6) to provide2-[[(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,42S,43S,44R,45R,55R)-44,55-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-48,49,50,51,52-pentaoxo-67,68-dioxa-57-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraen-42-y]oxy]ethylN-methylcarbamate (I-75: 18 mg, 15% yield) and2-[[(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,42R,43S,44R,45R,55R)-44,55-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-48,49,50,51,52-pentaoxo-67,68-dioxa-57-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraen-42-yl]oxy]ethylN-methylcarbamate (I-74: 20 mg, 16% yield), both as white solids.

Chiral Separation Method:

Column: CHIRALPAK IC

Column size: 5.0 cm I.D.×25 cm L

Solution concentration: 2.4 mg/ml

Injection: 8 ml

Mobile phase: Hexane/EtOH=60/40(V/V)

Flow rate: 30 ml/min

Wave length: UV 254 nm

Temperature: 35° C.

I-75: ESI-MS (EI⁺, m/z): 1067.3 [M+Na]⁺. ¹H NMR (400 MHz, CDCl3) δ 6.33(dt, J=24.4, 14.6 Hz, 2H), 6.13 (dd, J=15.0, 9.9 Hz, 1H), 5.93 (dd,J=24.5, 10.6 Hz, 1H), 5.52 (dd, J=15.0, 8.8 Hz, 1H), 5.41 (d, J=10.2 Hz,1H), 5.35 (t, J=4.7 Hz, 1H), 5.27 (d, J=5.3 Hz, 1H), 5.16 (d, J=4.9 Hz,1H), 4.75 (s, 1H), 4.29-4.07 (m, 3H), 3.93-3.63 (m, 6H), 3.62-3.47 (m,3H), 3.46-3.25 (m, 11H), 3.23-3.01 (m, 3H), 2.79 (d, J=4.9 Hz, 3H),2.74-2.62 (m, 2H), 2.57 (dd, J=16.5, 6.4 Hz, 1H), 2.34 (d, J=12.7 Hz,2H), 2.25-2.19 (m, 1H), 2.08 (s, 1H), 1.93 (dd, J=30.0, 22.7 Hz, 5H),1.83-1.65 (m, 7H), 1.55-1.42 (m, 5H), 1.28 (s, 6H), 1.15-0.83 (m, 18H),0.72 (dd, J=23.6, 12.0 Hz, 1H).

I-74: ESI-MS (EI⁺, m/z): 1067.4 [M+Na]⁺. ¹H NMR (400 MHz, CDCl3) δ6.50-5.95 (m, 4H), 5.60-4.96 (m, 5H), 4.59-3.96 (m, 4H), 3.95-3.66 (m,6H), 3.64-2.97 (m, 15H), 2.95-2.65 (m, 6H), 2.59 (d, J=11.0 Hz, 1H),2.51-1.95 (m, 5H), 1.78 (q, J=6.8 Hz, 12H), 1.55-1.29 (m, 11H),1.15-0.83 (m, 18H), 0.69 (dd, J=23.6, 11.5 Hz, 1H).

Example 33: Synthesis of(21E,23E,25E,26E,30R,31S,32R,33R,35S,37S,40S,41R,42R,55R)-41,55-dihydroxy-40-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-42-methoxy-30,31,32,33,43,44-hexamethyl-39-[(2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexoxy]-70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(43),26(44)-tetraene-45,46,47,48,49-pentone(I-63)and(21E,23E,25E,26E,30R,31S,32R,33R,35S,37S,39R,40S,41R,42R,55R)-41,55-dihydroxy-40-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-42-methoxy-30,31,32,33,43,44-hexamethyl-39-[(2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexoxy]-70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(43),26(44)-tetraene-45,46,47,48,49-pentone(I-57) and(21E,23E,25E,26E,30R,31S,32R,33R,35S,37S,39S,40S,41R,42R,55R)-41,55-dihydroxy-40-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-42-methoxy-30,31,32,33,43,44-hexamethyl-39-[(2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexoxy]-70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(43),26(44)-tetraene-45,46,47,48,49-pentone(I-58)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of(2R,3R,4R,5R)-2,3,4,5-tetrahydroxyhexane-1,6-diyl dibenzoate

To a solution of (2R,3R,4R,5R)-hexane-1,2,3,4,5,6-hexaol (10.0 g, 54.89mmol, 1.0 eq) in pyridine (25.39 g, 548.9 mmol, 10.0 eq) at 0° C. wasadded benzyl chloride (7.72 g, 54.89 mmol, 1.0 eq). The reaction mixturewas then stirred at room temperature for 16 h, diluted with H₂O (200 mL)and extracted with DCM (150 mL×3). The combined organic layers werewashed with brine (100 mL), dried over Na₂SO₄, filtered, concentrated,and purified by reverse-phase chromatography (CH₃CN:H₂O=40%˜60% yield)to afford (2R,3R,4R,5R)-2,3,4,5-tetrahydroxyhexane-1,6-diyl dibenzoate(4.7 g, 22% yield) as a white solid. ESI-MS (EI⁺, m/z): 391.1 [M+H]⁺.HNMR (400 MHz, DMSO-d₆) δ 8.07-8.00 (m, 4H), 7.66 (t, J=7.4 Hz, 2H),7.54 (t, J=7.7 Hz, 4H), 5.09 (d, J=6.1 Hz, 2H), 4.60-4.48 (m, 4H), 4.28(dd, J=11.2, 6.2 Hz, 2H), 3.87 (dt, J=6.1, 5.3 Hz, 2H), 3.77 (t, J=8.6Hz, 2H).

Step 2: Synthesis of((4R,4′R,5R,5′R)-2,2,2′,2′-tetramethyl-4,4′-bi(1,3-dioxolane)-5,5′-diyl)bis(methylene)dibenzoate and((4R,4aR,8R,8aR)-2,2,6,6-tetramethyltetrahydro-[1,3]dioxino[5,4-d][1,3]dioxine-4,8-diyl)bis(methylene)dibenzoate

A solution of [(2R,3R,4R,5R)-6-benzoyloxy-2,3,4,5-tetrahydroxy-hexyl]benzoate (5 g, 12.81 mmol) and p-TsOH (1.22 g, 6.40 mmol) in2,2-dimethoxypropane (50 mL) was stirred at 28° C. for 10 h. Theresulting mixture was extracted with EtOAc (100 mL×3). The combinedorganic layers were dried over anhydrous Na₂SO₄, filtered andconcentrated. The residue was purified by silica gel chromatography(EtOAc:PE=1:10 to 1:2) to provide both[(4R,5R)-5-[(4R,5R)-5-(benzoyloxymethyl)-2,2-dimethyl-1,3-dioxolan-4-yl]-2,2-dimethyl-1,3-dioxolan-4-yl]methylbenzoate (2.4 g, 40% yield) and[(4R,4aR,8R,8aR)-4-(benzoyloxymethyl)-2,2,6,6-tetramethyl-4,4a,8,8a-tetrahydro-[1,3]dioxino[5,4-d][1,3]dioxin-8-yl]methylbenzoate 1.2 g, 20% yield) as white solids. ESI-MS (EI⁺, m/z): 493.2[M+Na]⁺.

[(4R,5R)-5-[(4R,5R)-5-(benzoyloxymethyl)-2,2-dimethyl-1,3-dioxolan-4-yl]-2,2-dimethyl-1,3-dioxolan-4-yl]methylbenzoate: ¹HNMR (400 MHz, DMSO-d₆): δ 7.98 (dd, J=11.6, 4.5 Hz, 4H),7.67 (dd, J=11.7, 4.3 Hz, 2H), 7.55 (t, J=7.7 Hz, 4H), 4.42-4.33 (m,4H), 4.08-3.94 (m, 4H), 1.33 (s, 6H), 1.26 (s, 6H). ¹³C NMR (101 MHz,DMSO-d₆) δ 165.52, 133.44, 129.47, 129.15, 128.78, 100.61, 67.89, 67.60,64.25, 24.24, 23.45.

[(4R,4aR,8R,8aR)-4-(benzoyloxymethyl)-2,2,6,6-tetramethyl-4,4a,8,8a-tetrahydro-[1,3]dioxino[5,4-d][1,3]dioxin-8-yl]methylbenzoate: ¹HNMR (400 MHz, DMSO-d₆): δ 8.03-7.92 (m, 4H), 7.67 (dd,J=10.6, 4.3 Hz, 2H), 7.54 (t, J=7.7 Hz, 4H), 4.57 (dd, J=11.1, 5.9 Hz,2H), 4.53-4.44 (m, 4H), 4.39 (dd, J=11.5, 7.3 Hz, 2H), 1.43 (s, 6H),1.29 (s, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 165.45, 133.42, 129.52,129.17, 128.75, 108.39, 74.49, 73.77, 64.17, 27.04, 25.27.

Step 3: Synthesis of((4R,4′R,5R,5′R)-2,2,2′,2′-tetramethyl-4,4′-bi(1,3-dioxolane)-5,5′-diyl)dimethanol

A mixture of[(4R,5R)-5-[(4R,5R)-5-(benzoyloxymethyl)-2,2-dimethyl-1,3-dioxolan-4-yl]-2,2-dimethyl-1,3-dioxolan-4-yl]methylbenzoate (8.5 g, 18.07 mmol) and K₂CO₃ (7.48 g, 54.20 mmol) in THF (25mL) and CH₃OH (25 mL) was stirred at 29° C. for 18 h. The reaction wasconcentrated and then purified via silica gel chromatography(EtOAc/PE=1:1) to afford[(4R,5R)-5-[(4R,5R)-5-(hydroxymethyl)-2,2-dimethyl-1,3-dioxolan-4-yl]-2,2-dimethyl-1,3-dioxolan-4-yl]methanol(3.2 g, 68% yield) as a white solid. ESI-MS (EI⁺, m/z): 285.1 [M+Na]⁺.¹H NMR (400 MHz, DMSO-d₆): δ 4.73 (t, J=5.8 Hz, 2H), 3.71 (t, J=5.6 Hz,2H), 3.55-3.35 (m, 6H), 1.29 (s, 6H), 1.23 (s, 6H).

Step 4: Synthesis of(25E,27E,29E,30E,34R,35S,36R,37R,39S,41S,44S,49R,50R,61R)-49,61-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-43-[[(4R,5R)-5-[(4R,5R)-5-(hydroxymethyl)-2,2-dimethyl-1,3-dioxolan-4-yl]-2,2-dimethyl-1,3-dioxolan-4-yl]methoxy]-50-methoxy-34,35,36,37,51,52-hexamethyl-72,73-dioxa-62-azatricyclohexatriaconta-25,27,29(51),30(52)-tetraene-53,54,55,56,57-pentone

To a solution of rapamycin (0.5 g, 0.547 mol) in DCM (16 mL) was added2,2,2-trifluoroacetic acid (1.2 mL) at −40° C. After stirring for 10minutes[(4R,5R)-5-[(4R,5R)-5-(hydroxymethyl)-2,2-dimethyl-1,3-dioxolan-4-yl]-2,2-dimethyl-1,3-dioxolan-4-yl]methanol(0.43 g, 1.64 mmol) was added and the reaction was stirred at −30° C.for 1.5 h, then diluted with DCM (10 mL) and poured into a cold aqueousNaHCO₃ solution. The organic layer was washed with water and brine,dried over anhydrous Na₂SO₄, filtered and concentrated. The resultingresidue was purified via reverse phase chromatography (C18,CH₃CN/H₂O=7:3) to provide(25E,27E,29E,30E,34R,35S,36R,37R,39S,41S,44S,49R,50R,61R)-49,61-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-43-[[(4R,5R)-5-[(4R,5R)-5-(hydroxymethyl)-2,2-dimethyl-1,3-dioxolan-4-yl]-2,2-dimethyl-1,3-dioxolan-4-yl]methoxy]-50-methoxy-34,35,36,37,51,52-hexamethyl-72,73-dioxa-62-azatricyclohexatriaconta-25,27,29(51),30(52)-tetraene-53,54,55,56,57-pentone(150 mg, 24% yield) as a white solid. ESI-MS (EI⁺, m/z): 1166.4 [M+Na]⁺.¹HNMR (400 MHz, CDCl₃) δ 6.52-5.69 (m, 4H), 5.48-5.10 (m, 4H), 4.51 (d,J=40.1 Hz, 1H), 4.23 (s, 1H), 3.94 (dd, J=26.3, 5.8 Hz, 4H), 3.52-3.12(m, 13H), 2.98-2.45 (m, 8H), 2.41-2.17 (m, 2H), 2.00 (s, 3H), 1.76 (dt,J=29.0, 14.4 Hz, 15H), 1.50-1.31 (m, 24H), 1.14-0.77 (m, 18H), 0.62 (d,J=12.1 Hz, 1H).

Step 5: Synthesis of(21E,23E,25E,26E,30R,31S,32R,33R,35S,37S,40S,41R,42R,55R)-41,55-dihydroxy-40-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-42-methoxy-30,31,32,33,43,44-hexamethyl-39-[(2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexoxyl]-70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(43),26(44)-tetraene-45,46,47,48,49-pentone(I-63)

To a solution of(25E,27E,29E,30E,34R,35S,36R,37R,39S,41S,44S,49R,50R,61R)-49,61-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-43-[[(4R,5R)-5-[(4R,5R)-5-(hydroxymethyl)-2,2-dimethyl-1,3-dioxolan-4-yl]-2,2-dimethyl-1,3-dioxolan-4-yl]methoxy]-50-methoxy-34,35,36,37,51,52-hexamethyl-72,73-dioxa-62-azatricyclohexatriaconta-25,27,29(51),30(52)-tetraene-53,54,55,56,57-pentone(500 mg, 0.437 mmol) in 1,4-dioxane (6 mL) and H₂O (6 mL) was addedDowex 50W-X8 (1.0 g) and the resulting mixture stirred at 50° C. for 24h, then filtered and purified by reverse phase chromatography (C18,CH₃CN:H₂O=4:6) to provide(21E,23E,25E,26E,30R,31S,32R,33R,35S,37S,40S,41R,42R,55R)-41,55-dihydroxy-40-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-42-methoxy-30,31,32,33,43,44-hexamethyl-39-[(2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexoxy]-70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(43),26(44)-tetraene-45,46,47,48,49-pentone(I-63: 70 mg, 15% yield) as a white solid. ESI-MS (EI⁺, m/z): 1086.4[M+Na]⁺. ¹H NMR (400 MHz, CDCl3) δ 6.51-5.91 (m, 4H), 5.61-4.98 (m, 5H),4.24 (d, J=22.2 Hz, 1H), 3.78 (dd, J=34.1, 28.6 Hz, 9H), 3.59-3.17 (m,19H), 3.00-2.49 (m, 5H), 2.38-2.22 (m, 1H), 2.05 (d, J=33.7 Hz, 3H),1.82-1.68 (m, 11H), 1.29 (ddd, J=46.4, 34.9, 8.8 Hz, 12H), 1.09-0.77 (m,18H), 0.70-0.56 (m, 1H).

Step 6: Synthesis of(21E,23E,25E,26E,30R,31S,32R,33R,35S,37S,39S,40S,41R,42R,55R)-41,55-dihydroxy-40-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-42-methoxy-30,31,32,33,43,44-hexamethyl-39-[(2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexoxy]-70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(43),26(44)-tetraene-45,46,47,48,49-pentone(I-58) and(21E,23E,25E,26E,30R,31S,32R,33R,35S,37S,39R,40S,41R,42R,55R)-41,55-dihydroxy-40-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-42-methoxy-30,31,32,33,43,44-hexamethyl-39-[(2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexoxy]-70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(43),26(44)-tetraene-45,46,47,48,49-pentone(I-57)

90 mg of(21E,23E,25E,26E,30R,31S,32R,33R,35S,37S,40S,41R,42R,55R)-41,55-dihydroxy-40-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-42-methoxy-30,31,32,33,43,44-hexamethyl-39-[(2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexoxy]-70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(43),26(44)-tetraene-45,46,47,48,49-pentonewas purified via prep chiral HPLC and the resulting epimers purified viasilica gel chromatography (hexane:DCM:EtOAc:MeOH=3:3:1:1) to provide(21E,23E,25E,26E,30R,31S,32R,33R,35S,37S,39S,40S,41R,42R,55R)-41,55-dihydroxy-40-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-42-methoxy-30,31,32,33,43,44-hexamethyl-39-[(2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexoxy]-70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(43),26(44)-tetraene-45,46,47,48,49-pentone(I-58: 15 mg, 17%) and(21E,23E,25E,26E,30R,31S,32R,33R,35S,37S,39R,40S,41R,42R,55R)-41,55-dihydroxy-40-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-42-methoxy-30,31,32,33,43,44-hexamethyl-39-[(2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexoxy]-70,71-dioxa-56-azatricyclohexatriaconta-21,23,25(43),26(44)-tetraene-45,46,47,48,49-pentone(I-57: 8 mg, 9% yield), both as white solids.

Chiral Analysis Method:

Column: CHIRALPAK IC-3(IC30CE-NJ008)

Column size: 0.46 cm I.D.×25 cm L

Injection: 50.0 ul

Mobile phase: Hexane/EtOH=50/50(V/V)

Flow rate: 0.8 ml/min

Wave length: UV 254 nm

Temperature: 35° C.

HPLC equipment: Shimadzu LC-20AT CP-HPLC-06

I-58: ESI-MS (EI⁺, m/z): 1086.3 [M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ6.45-5.93 (m, 4H), 5.59-4.88 (m, 5H), 4.23 (d, J=26.4 Hz, 1H), 4.00-3.48(m, 14H), 3.46-3.23 (m, 11H), 2.93 (d, J=6.8 Hz, 2H), 2.79-2.49 (m, 3H),2.38-1.84 (m, 8H), 1.66 (t, J=14.8 Hz, 9H), 1.50-1.17 (m, 11H),1.16-0.79 (m, 18H), 0.64 (dd, J=23.7, 11.8 Hz, 1H).

I-57: ESI-MS (EI⁺, m/z): 1086.3 [M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ6.49-5.98 (m, 4H), 5.52-5.11 (m, 5H), 4.32-4.23 (m, 1H), 4.08-3.11 (m,14H), 3.05-2.43 (m, 9H), 2.37-1.95 (m, 8H), 1.91-1.53 (m, 17H),1.46-0.54 (m, 30H).

Example 34: Synthesis of(21E,23E,25E,26E,40R,41S,42R,43R,45S,47S,50S,51R,52R,61R)-51,61-dihydroxy-49-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-50-[(1R)-2-[(1S,3R,4R)-4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-52-methoxy-40,41,42,43,53,54-hexamethyl-72,73-dioxa-62-azatricyclohexatriaconta-21,23,25(53),26(54)-tetraene-55,56,57,58,59-pentone(I-78) and(21E,23E,25E,26E,40R,41S,42R,43R,45S,47S,49R,50S,51R,52R,61R)-51,61-dihydroxy-49-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-50-[(1R)-2-[(1S,3R,4R)-4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-52-methoxy-40,41,42,43,53,54-hexamethyl-72,73-dioxa-62-azatricyclohexatriaconta-21,23,25(53),26(54)-tetraene-55,56,57,58,59-pentone(I-72) and(21E,23E,25E,26E,40R,41S,42R,43R,45S,47S,49S,50S,51R,52R,61R)-51,61-dihydroxy-49-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-50-[(1R)-2-[(1S,3R,4R)-4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-52-methoxy-40,41,42,43,53,54-hexamethyl-72,73-dioxa-62-azatricyclohexatriaconta-21,23,25(53),26(54)-tetraene-55,56,57,58,59-pentone(I-73)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethanol

To a solution of 2-(2-hydroxyethoxy)ethanol (50 g, 471.2 mmol) inpyridine (49.6 mL) at 0° C. was added tert-butyl-chloro-diphenyl-silane(30 g, 109.2 mmol). The resulting solution was stirred at rt for 1h thenpoured into water (300 mL) and extracted with EtOAc (300 mL×3). Thecombined organic layers were dried over anhydrous Na₂SO₄, filtered, andconcentrated. The resulting residue was purified by silica gelchromatography (EtOAc:PE=1:8) to provide2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethanol (29.9 g, 80% yield) asa colorless oil. ESI-MS (EI⁺, m/z): 367.2 [M+Na]⁺. ¹H NMR (400 MHz,CDCl₃) δ 7.74-7.64 (m, 4H), 7.46-7.33 (m, 6H), 3.81 (t, J=5.2 Hz, 2H),3.73-3.66 (m, 2H), 3.63-3.54 (m, 4H), 2.32 (d, J=3.5 Hz, 1H), 1.06 (s,9H).

Step 2: Synthesis of 2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethylTrifluoromethanesulfonate

To a solution of 2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethanol (7.6g, 22 mmol) and DIPEA (5.76 mL) in DCM (50 mL) at 0° C., under N₂, wasadded trifluoromethylsulfonyl 1,1-difluoroethanesulfonate (3.92 mL) Themixture was then diluted with DCM (150 mL), washed with saturated NaHCO₃(150 mL), water (150 mL) and brine (150 mL). The organic layer was thendried over anhydrous Na₂SO₄, filtered and concentrated under vacuum toprovide 2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethyltrifluoromethanesulfonate (10.21 g, 97% yield) as a brown oil which wasused without any further purification. ¹H NMR (400 MHz, CDCl₃) δ7.69-7.66 (m, 4H), 7.43-7.36 (m, 6H), 4.58 (t, J=4.8 Hz, 2H), 3.83-3.80(m, 4H), 3.64-3.61 (t, J=5.6 Hz, 2H), 1.05 (s, 9H).

Step 3: Synthesis of(35E,37E,39E,40E,50R,51S,52R,53R,55S,57S,59S,60S,61R,62R,71R)-60-[(1R)-2-[(1S,3R,4R)-4-[2-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-61,71-dihydroxy-59,62-dimethoxy-50,51,52,53,63,64-hexamethyl-81,82-dioxa-73-azatricyclohexatriaconta-35,37,39(63),40(64)-tetraene-65,66,67,68,69-pentone

A mixture of everolimus (2 g, 2.09 mmol),2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethyltrifluoromethanesulfonate (9.95 g, 20.87 mmol) andN-ethyl-N-isopropyl-propan-2-amine (5.82 mL) in toluene (50 mL) wasstirred at 60° C. for 18 h then poured into ice cold sat.NaHCO₃(60 mL).The reaction mixture was extracted with EtOAc (40 mL) and the organiclayer washed with water (50 mL×3) and brine (50 mL) then dried overanhydrous Na₂SO₄, filtered and concentrated. The residue was purified bysilica gel chromatography (PE:EA=5:1 to 3:1) to provide(35E,37E,39E,40E,50R,51S,52R,53R,55S,57S,59S,60S,61R,62R,71R)-60-[(1R)-2-[(1S,3R,4R)-4-[2-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-61,71-dihydroxy-59,62-dimethoxy-50,51,52,53,63,64-hexamethyl-81,82-dioxa-73-azatricyclohexatriaconta-35,37,39(63),40(64)-tetraene-65,66,67,68,69-pentone(1.7 g, 63% yield) as yellow solid. ESI-MS (EI⁺, m/z): 1307.5 [M+Na]⁺.

Step 4: Synthesis of(22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,44S,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-44,47-dimethoxy-35,36,37,38,48,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone

To a solution of(35E,37E,39E,40E,50R,51S,52R,53R,55S,57S,59S,60S,61R,62R,71R)-60-[(1R)-2-[(1S,3R,4R)-4-[2-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-61,71-dihydroxy-59,62-dimethoxy-50,51,52,53,63,64-hexamethyl-81,82-dioxa-73-azatricyclohexatriaconta-35,37,39(63),40(64)-tetraene-65,66,67,68,69-pentone(322 mg, 0.251 mmol) in THF (10 mL) was added HF pyridine (248.5 mg,2.51 mmol). The resulting solution was stirred at rt for 3h, then pouredinto saturated aqueous NaHCO₃ and extracted with EtOAc. The organiclayer was concentrated and purified via silica gel chromatography(acetone:PE=1:3) to provide(22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,44S,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-44,47-dimethoxy-35,36,37,38,48,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone(114 mg, 44% yield) as light yellow solid. ESI-MS (EI⁺, m/z): 1069.3[M+Na]⁺.

Step 5: Synthesis of(21E,23E,25E,26E,40R,41S,42R,43R,45S,47S,50S,51R,52R,61R)-51,61-dihydroxy-49-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-50-[(1R)-2-[(1S,3R,4R)-4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-52-methoxy-40,41,42,43,53,54-hexamethyl-72,73-dioxa-62-azatricyclohexatriaconta-21,23,25(53),26(54)-tetraene-55,56,57,58,59-pentone(I-78)

A mixture of(22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,44S,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-44,47-dimethoxy-35,36,37,38,48,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone(0.95 g, 0.908 mmol), 2-[2-(2-hydroxyethoxy)ethoxy]ethanol (2 mL) andp-toluenesulfonic acid (0.78 g, 4.54 mmol) in THF (20 mL) was stirred at20° C. for 2 h. The reaction was poured into ice-cold saturated aqueousNaHCO₃ (30 mL) and extracted with EtOAc (50 mL×3). The combined organiclayers were washed with water and brine, then concentrated and purifiedvia reverse-phase chromatography (C18, CH₃CN:H₂O=6.5:1) to provide(21E,23E,25E,26E,40R,41S,42R,43R,45S,47S,50S,51R,52R,61R)-51,61-dihydroxy-49-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-50-[(1R)-2-[(1S,3R,4R)-4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-52-methoxy-40,41,42,43,53,54-hexamethyl-72,73-dioxa-62-azatricyclohexatriaconta-21,23,25(53),26(54)-tetraene-55,56,57,58,59-pentone(I-78: 0.317 g, 30% yield) as a white solid. ESI-MS (EI⁺, m/z): 1186.4[M+Na]⁺. ¹H NMR (400 MHz, CDCl3) δ 6.39-5.95 (m, 4H), 5.59-5.34 (m, 2H),5.26-5.09 (m, 2H), 4.81 (s, 1H), 4.29-4.15 (m, 1H), 4.00-3.49 (m, 28H),3.48-3.34 (m, 9H), 3.16-3.0 (m, 4H), 2.80-2.52 (m, 3H), 2.34-2.20 (m,2H), 2.07-1.88 (m, 4H), 1.79-1.72 (m, 5H), 1.66 (s, 3H), 1.51-1.38 (m,4H), 1.37-1.22 (m, 7H), 1.21-0.84 (m, 20H), 0.76-0.64 (m, 1H).

Step 6: Synthesis of(21E,23E,25E,26E,40R,41S,42R,43R,45S,47S,49S,50S,51R,52R,61R)-51,61-dihydroxy-49-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-50-[(1R)-2-[(1S,3R,4R)-4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-52-methoxy-40,41,42,43,53,54-hexamethyl-72,73-dioxa-62-azatricyclohexatriaconta-21,23,25(53),26(54)-tetraene-55,56,57,58,59-pentone(I-73) and(21E,23E,25E,26E,40R,41S,42R,43R,45S,47S,49R,50S,51R,52R,61R)-51,61-dihydroxy-49-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-50-[(1R)-2-[(1S,3R,4R)-4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-52-methoxy-40,41,42,43,53,54-hexamethyl-72,73-dioxa-62-azatricyclohexatriaconta-21,23,25(53),26(54)-tetraene-55,56,57,58,59-pentone(I-72)

330 mg of(21E,23E,25E,26E,40R,41S,42R,43R,45S,47S,50S,51R,52R,61R)-51,61-dihydroxy-49-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-50-[(1R)-2-[(1S,3R,4R)-4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-52-methoxy-40,41,42,43,53,54-hexamethyl-72,73-dioxa-62-azatricyclohexatriaconta-21,23,25(53),26(54)-tetraene-55,56,57,58,59-pentonewas purified via prep chiral HPLC and the resulting epimers purified viasilica gel chromatography (hexane:DCM:EtOAc:MeOH=3:3:1:1) to provide(21E,23E,25E,26E,40R,41S,42R,43R,45S,47S,49S,50S,51R,52R,61R)-51,61-dihydroxy-49-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-50-[(1R)-2-[(1S,3R,4R)-4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-52-methoxy-40,41,42,43,53,54-hexamethyl-72,73-dioxa-62-azatricyclohexatriaconta-21,23,25(53),26(54)-tetraene-55,56,57,58,59-pentone(I-73: 77 mg, 23% yield) and(21E,23E,25E,26E,40R,41S,42R,43R,45S,47S,49R,50S,51R,52R,61R)-51,61-dihydroxy-49-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-50-[(1R)-2-[(1S,3R,4R)-4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-52-methoxy-40,41,42,43,53,54-hexamethyl-72,73-dioxa-62-azatricyclohexatriaconta-21,23,25(53),26(54)-tetraene-55,56,57,58,59-pentone(I-72: 50 mg, 15% yield), both as white solids.

Chiral Separation Method:

Column: CHIRALPAK IC

Column size: 2.5 cm I.D.×25 cm L

Solution concentration: 6.5 mg/ml

Injection: 7 ml

Mobile phase: Hexane/EtOH=60/40 (V/V)

Flow rate: 40 ml/min

Wave length: UV 254 nm

Temperature: 35° C.

I-73: ESI-MS (EI⁺, m/z): 1186.6 [M+Na]⁺. ¹H NMR (400 MHz, CDCl3) δ6.42-6.06 (m, 3H), 5.92 (dd, J=29.5, 10.8 Hz, 1H), 5.55-5.37 (m, 2H),5.26 (d, J=5.2 Hz, 1H), 5.14 (d, J=4.3 Hz, 1H), 4.80 (s, 1H), 4.23 (d,J=24.9 Hz, 1H), 3.90 (s, 1H), 3.81-3.22 (m, 36H), 3.19-2.99 (m, 3H),2.78-2.48 (m, 3H), 2.33 (d, J=12.4 Hz, 2H), 2.10-1.87 (m, 5H), 1.76-1.55(m, 13H), 1.46 (s, 4H), 1.39-1.18 (m, 5H), 1.15-0.81 (m, 18H), 0.71 (dd,J=23.5, 11.7 Hz, 1H).

I-72: ¹H NMR (400 MHz, CDCl₃) δ 6.42-5.92 (m, 4H), 5.61-5.04 (m, 5H),4.24 (d, J=53.9 Hz, 2H), 3.99 (dd, J=13.8, 6.9 Hz, 1H), 3.83-2.90 (m,36H), 2.75-2.46 (m, 3H), 2.16 (ddd, J=109.4, 53.2, 24.9 Hz, 7H),1.86-1.69 (m, 7H), 1.52-1.16 (m, 17H), 1.15-0.80 (m, 18H), 0.66 (dd,J=23.9, 11.6 Hz, 1H).

Example 35: Synthesis of(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone(I-91)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxymethylbenzene

To a slurry of sodium hydride (12.49 g, 520.5 mmol) in DMF (150 mL) wasadded 2-(2,2,2-trifluoroethoxy)ethanol (5 g, 34.7 mmol) in DMF (10 mL)under N₂ atmosphere at 0° C. The mixture was stirred for 1h then2-bromoethoxymethylbenzene (18.66 g, 86.75 mmol) was added dropwise. Themixture was stirred at room temperature for 20 h then quenched withwater (50 mL) and extracted with EtOAc (80 mL). The organic layer waswashed with water (50 mL×3), brine (50 mL), dried over anhydrous Na₂SO₄,filtered, and concentrated. The residue was purified via silica gelchromatography (PE:EA=25:1 to 20:1) to provide2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxymethylbenzene (8.1 g, 84%yield) as a colorless liquid. ¹H NMR (400 MHz, CDCl₃): δ 7.37-7.26 (m,5H), 4.57 (s, 2H), 3.90 (q, J=8.8 Hz, 2H), 3.79 (dd, J=5.6, 3.5 Hz, 2H),3.71-3.61 (m, 6H).

Step 2: Synthesis of 2-[2-(2,2,2-trifluoroethoxy)ethoxy]

To a solution of 2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxymethylbenzene(0.5 g, 1.8 mmol) in CH₃OH (10 mL) was added Pd/C (0.43 g) and thereaction stirred under H₂ atmosphere at room temperature (20° C.) for 20h. The Pd/C was removed via filtration and the resulting filtrateconcentrated and purified by silica gel chromatography (DCM:CH₃OH=50:1)to provide 2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethanol (0.30 g, 89%yield) as colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 3.91 (q, J=8.7 Hz,2H), 3.80 (dd, J=5.6, 3.4 Hz, 2H), 3.75 (d, J=4.0 Hz, 2H), 3.70 (dd,J=5.5, 3.5 Hz, 2H), 3.62 (dd, J=5.2, 3.9 Hz, 2H), 2.23 (t, J=5.7 Hz, 1H(OH)). ¹⁹F NMR (376 MHz, CDCl₃, (trifluoromethylbenzene as standard)8-74.33 (t, J=8.8 Hz).

Step 3: Synthesis of(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone

To a degassed solution of(22E,24E,26E,27E,29R,30S,31R,32R,34S,36S,38S,39S,40R,41R,50R)-40,50-dihydroxy-39-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-38,41-dimethoxy-29,30,31,32,42,43-hexamethyl-60,61-dioxa-51-azatricyclohexatriaconta-22,24,26(42),27(43)-tetraene-44,45,46,47,48-pentone(0.1 g, 0.11 mmol) in THF (5 mL) at 0° C. was added p-toluenesulfonicacid (94 mg, 0.547 mmol) and 2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethanol(0.2 g, 1.09 mmol). The resulting mixture was stirred at 23° C. for 5 hunder N₂ then poured into sat.NaHCO₃(40 mL) then extracted with EtOAc(30 mL). The organic layer was washed with water (30 mL×2), brine (20mL), dried over anhydrous Na₂SO₄, filtered, and concentrated. Theresidue was purified by reverse phase chromatography (C₁₈, CH₃CN:H₂O=0%to 65% yield) to provide(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone(I-91: 30 mg, 26% yield) as a white solid. ESI-MS (EI⁺, m/z): 1093.5[M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ 6.38-5.90 (m, 4H), 5.46-5.05 (m, 4H),4.5-4.4 (m, 1H), 4.21-4.11 (m, 2H), 3.91-3.52 (m, 6H), 3.34-3.25 (m,8H), 3.07 (s, 1H), 2.84-2.59 (m, 5H), 2.31-1.91 (m, 6H), 1.77-1.54 (m,22H), 1.43-1.19 (m, 10H), 1.04-0.80 (m, 16H), 0.60 (q, J=12 Hz, 1H).

Example 36: Synthesis of(23E,25E,27E,28E,36R,37S,38R,39R,41S,43S,46S,47R,48R,57R)-47,57-dihydroxy-48-methoxy-45-[2-(2-methoxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1-methyl-ethyl]-36,37,38,39,49,50-hexamethyl-66,67-dioxa-58-azatricyclohexatriaconta-23,25,27(49),28(50)-tetraene-51,52,53,54,55-pentone(I-92) and(23E,25E,27E,28E,36R,37S,38R,39R,41S,43S,45S,46S,47R,48R,57R)-47,57-dihydroxy-48-methoxy-45-[2-(2-methoxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1-methyl-ethyl]-36,37,38,39,49,50-hexamethyl-66,67-dioxa-58-azatricyclohexatriaconta-23,25,27(49),28(50)-tetraene-51,52,53,54,55-pentone(I-90) and(23E,25E,27E,28E,36R,37S,38R,39R,41S,43S,45R,46S,47R,48R,57R)-47,57-dihydroxy-48-methoxy-45-[2-(2-methoxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1-methyl-ethyl]-36,37,38,39,49,50-hexamethyl-66,67-dioxa-58-azatricyclohexatriaconta-23,25,27(49),28(50)-tetraene-51,52,53,54,55-pentone(I-89)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of 2-methoxyethylTrifluoromethanesulfonate

A solution of 2-methoxyethanol (3 g, 39.42 mmol) and DIPEA (10.30 mL,59.14 mmol) in DCM (60 mL) was cooled to 0° C. under N₂, andtrifluoromethanesufonic anhydride (7.28 mL, 43.37 mmol,) added dropwise.The mixture was stirred at 0° C. for 2 h then diluted with DCM (50 mL).The organic layer was washed with sat. NaHCO₃ (50 mL), water (50 mL),brine (50 mL), then dried over anhydrous Na₂SO₄, filtrated, andconcentrated under vacuum to afford 2-methoxyethyltrifluoromethanesulfonate as brown oil. This was used in the next stepwithout further purification. ¹H NMR (400 MHz, CDCl₃) δ 4.62-4.58 (t,J=4.4 Hz 2H), 3.70-3.65 (t, J=4.4 Hz, 2H), 3.39 (s, 3H).

Step 2: Synthesis of(23E,25E,27E,28E,32R,33S,34R,35R,37S,39S,41S,42S,43R,44R,53R)-43,53-dihydroxy-41,44-dimethoxy-42-[(1R)-2-[(1S,3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1-methyl-ethyl]-32,33,34,35,45,46-hexamethyl-62,63-dioxa-54-azatricyclohexatriaconta-23,25,27(45),28(46)-tetraene-47,48,49,50,51-pentone

A mixture of rapamycin (2 g, 2.19 mmol), 2-methoxyethyltrifluoromethanesulfonate and N-ethyl-N-isopropyl-propan-2-amine (6.48mL, 37.19 mmol) in toluene (60 mL) was stirred at 58° C. for 18 h thendiluted with EtOAc (100 mL), poured into ice-cold Sat. NaHCO₃ (150 mL),washed with ice-cold water twice (250 mL), brine (200 mL), dried overanhydrous Na₂SO₄, filtered and concentrated. The crude was purified viasilica gel chromatography (acetone:PE=1:6) to provide(23E,25E,27E,28E,32R,33S,34R,35R,37S,39S,41S,42S,43R,44R,53R)-43,53-dihydroxy-41,44-dimethoxy-42-[(1R)-2-[(1S,3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1-methyl-ethyl]-32,33,34,35,45,46-hexamethyl-62,63-dioxa-54-azatricyclohexatriaconta-23,25,27(45),28(46)-tetraene-47,48,49,50,51-pentone(0.53 g, 25% yield) as a light brown solid. ESI-MS (EI⁺, m/z): 994.5[M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) (6.42-6.11 (m, 3H), 5.96 (m, 1H),5.58-5.40 (m, 2H), 5.29-5.14 (m, 2H), 4.80 (s, 1H), 4.18 (m, 1H),3.75-3.64 (m, 5H), 5.59-3.50 (m, 3H), 3.46-3.43 (m, 4H), 3.38-3.30 (m,6H), 3.17-3.05 (m, 4H), 2.80-2.56 (m, 3H), 2.06-1.92 (m, 4H), 1.86-1.75(m, 6H), 1.69-1.59 (m, 10H), 1.51-1.42 (m, 5H), 1.31-1.15 (m, 8H),1.11-1.04 (m, 6H), 1.00-0.83 (m, 10H), 0.72 (q, J=12 Hz, 1H).

Step 3: Synthesis of(23E,25E,27E,28E,36R,37S,38R,39R,41S,43S,46S,47R,48R,57R)-47,57-dihydroxy-48-methoxy-45-[2-(2-methoxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1-methyl-ethyl]-36,37,38,39,49,50-hexamethyl-66,67-dioxa-58-azatricyclohexatriaconta-23,25,27(49),28(50)-tetraene-51,52,53,54,55-pentone(I-92)

A solution of(23E,25E,27E,28E,32R,33S,34R,35R,37S,39S,41S,42S,43R,44R,53R)-43,53-dihydroxy-41,44-dimethoxy-42-[(1R)-2-[(1S,3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1-methyl-ethyl]-32,33,34,35,45,46-hexamethyl-62,63-dioxa-54-azatricyclohexatriaconta-23,25,27(45),28(46)-tetraene-47,48,49,50,51-pentone(0.45 g, 0.463 mmol) in THF (25 mL) was degassed with N₂,4-methylbenzenesulfonic acid (0.4 g, 2.31 mmol) was added at 0° C.,followed by 2-(2-methoxyethoxy)ethanol (4 mL). The resulting mixture wasstirred at 0° C. for 0.5 h under N₂, then at 25° C. for 3 h. The mixturewas poured into ice-cold sat. NaHCO₃ (50 mL), extracted with EtOAc (80mL×2), the organic layer was washed with water (100 mL), brine (100 mL)and concentrated under vacuum. The residue was purified via reversephase chromatography (C18, CH₃CN:H₂O from 10%˜75% yield) to afford(23E,25E,27E,28E,36R,37S,38R,39R,41S,43S,46S,47R,48R,57R)-47,57-dihydroxy-48-methoxy-45-[2-(2-methoxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1-methyl-ethyl]-36,37,38,39,49,50-hexamethyl-66,67-dioxa-58-azatricyclohexatriaconta-23,25,27(49),28(50)-tetraene-51,52,53,54,55-pentone(I-92: 0.11 g 22% yield) as a white solid. ESI-MS (EI⁺, m/z): 1082.5[M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ 6.32-5.99 (m, 3H), 5.89-5.78 (m, 1H),5.48-5.07 (m, 4H), 4.70-4.51 (m, 1H), 4.20-4.10 (m, 1H), 3.83-3.76 (m,1H), 3.73-3.62 (m, 3H), 3.57-5.43 (m, 8H), 3.41-3.37 (m, 4H), 3.35-3.21(m, 12H), 3.14-3.97 (m, 3H), 2.68-2.45 (m, 3H), 2.24 (m, 2H), 1.99-1.81(m, 4H), 1.68-1.52 (m, 15H), 1.44-1.34 (m, 4H), 1.26-1.14 (m, 5H),1.05-0.94 (m, 8H), 0.92-0.77 (m, 10H), 0.65 (q, J=12 Hz, 1H).

Step 4: Synthesis of(23E,25E,27E,28E,36R,37S,38R,39R,41S,43S,45S,46S,47R,48R,57R)-47,57-dihydroxy-48-methoxy-45-[2-(2-methoxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1-methyl-ethyl]-36,37,38,39,49,50-hexamethyl-66,67-dioxa-58-azatricyclohexatriaconta-23,25,27(49),28(50)-tetraene-51,52,53,54,55-pentone(I-90) and(23E,25E,27E,28E,36R,37S,38R,39R,41S,43S,45R,46S,47R,48R,57R)-47,57-dihydroxy-48-methoxy-45-[2-(2-methoxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1-methyl-ethyl]-36,37,38,39,49,50-hexamethyl-66,67-dioxa-58-azatricyclohexatriaconta-23,25,27(49),28(50)-tetraene-51,52,53,54,55-pentone(I-89)

1.16 g of(23E,25E,27E,28E,36R,37S,38R,39R,41S,43S,46S,47R,48R,57R)-47,57-dihydroxy-48-methoxy-45-[2-(2-methoxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1-methyl-ethyl]-36,37,38,39,49,50-hexamethyl-66,67-dioxa-58-azatricyclohexatriaconta-23,25,27(49),28(50)-tetraene-51,52,53,54,55-pentonewas purified via prep chiral HPLC and the resulting epimers purified viasilica gel chromatography (hexane: DCM:EtOAc:MeOH=3:3:1:0.3) to obtain(23E,25E,27E,28E,36R,37S,38R,39R,41S,43S,45S,46S,47R,48R,57R)-47,57-dihydroxy-48-methoxy-45-[2-(2-methoxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1-methyl-ethyl]-36,37,38,39,49,50-hexamethyl-66,67-dioxa-58-azatricyclohexatriaconta-23,25,27(49),28(50)-tetraene-51,52,53,54,55-pentone(I-90:340 mg, 28% yield) and(23E,25E,27E,28E,36R,37S,38R,39R,41S,43S,45R,46S,47R,48R,57R)-47,57-dihydroxy-48-methoxy-45-[2-(2-methoxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-3-methoxy-4-(2-methoxyethoxy)cyclohexyl]-1-methyl-ethyl]-36,37,38,39,49,50-hexamethyl-66,67-dioxa-58-azatricyclohexatriaconta-23,25,27(49),28(50)-tetraene-51,52,53,54,55-pentone(I-89: 135 mg, 11% yield), both as white solids.

Chiral Separation Method:

Column: CHIRALPAK IC

Column size: 2.5 cm I.D.×25 cm L

Solution concentration: 2.5 mg/ml

Injection: 3 ml

Mobile phase: Hexane/EtOH=60/40 (V/V)

Flow rate: 30 ml/min

Wave length: UV 254 nm

Temperature: 35° C.

I-90: ESI-MS (EI⁺, m/z): 1082.4 [M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ6.40-6.22 (m, 2H), 6.14 (dt, J=15.0, 9.8 Hz, 1H), 5.90 (dd, J=32.4, 10.7Hz, 1H), 5.56-5.32 (m, 2H), 5.27 (d, J=5.0 Hz, 1H), 5.16 (d, J=4.2 Hz,1H), 4.77 (s, 1H), 4.18 (d, J=5.9 Hz, 1H), 3.87 (s, 1H), 3.80-3.24 (m,29H), 3.22-3.01 (m, 3H), 2.72 (dd, J=16.9, 5.8 Hz, 2H), 2.57 (dd,J=16.8, 6.4 Hz, 1H), 2.30 (t, J=21.1 Hz, 2H), 1.93 (ddd, J=26.1, 21.2,9.8 Hz, 6H), 1.82-1.64 (m, 8H), 1.50 (dd, J=22.1, 10.9 Hz, 6H),1.37-1.15 (m, 6H), 1.15-0.82 (m, 18H), 0.71 (q, J=8.0, 20.0 Hz, 1H).

I-89: ESI-MS (EI⁺, m/z): 1082.4 [M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ6.41-5.93 (m, 4H), 5.59-5.37 (m, 2H), 5.20 (dd, J=23.8, 19.2 Hz, 2H),4.55 (d, J=10.4 Hz, 1H), 4.28 (s, 1H), 4.15 (d, J=10.2 Hz, 1H), 4.00 (d,J=3.7 Hz, 1H), 3.91-3.27 (m, 28H), 3.27-2.85 (m, 5H), 2.76-2.24 (m, 5H),2.18-1.55 (m, 14H), 1.54-1.20 (m, 10H), 1.16-0.82 (m, 18H), 0.75-0.61(m, 1H).

Example 37: Synthesis of(21E,23E,25E,26E,35R,36S,37R,38R,40S,42S,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-35,36,37,38,48,49-hexamethyl-44-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(48),26(49)-tetraene-50,51,52,53,54-pentone(I-86) and(21E,23E,25E,26E,35R,36S,37R,38R,40S,42S,44S,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-35,36,37,38,48,49-hexamethyl-44-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(48),26(49)-tetraene-50,51,52,53,54-pentone(I-85)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxymethylbenzene

To a slurry of sodium hydride (12.49 g, 520.5 mmol) in DMF (150 mL) wasadded 2-(2,2,2-trifluoroethoxy)ethanol (5 g, 34.7 mmol) in DMF (10 mL)under N₂ at 0° C. The mixture was stirred at for 0° C. 1 h, then2-bromoethoxymethylbenzene (18.66 g, 86.75 mmol) was added dropwise andthe reaction stirred at room temperature for 20 h. The mixture wasquenched by water (50 mL) and extracted with EtOAc (80 mL). The organiclayer was washed with water (50 mL×3), brine (50 mL), dried overanhydrous Na₂SO₄, filtered and concentrated. The residue was purifiedvia silica gel chromatography (PE:EtOAc=25:1 to 20:1) to obtain2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxymethylbenzene (8.1 g, 83.9%yield) as colorless liquid. ¹H NMR (400 MHz, CDCl₃): δ 7.37-7.26 (m,5H), 4.57 (s, 2H), 3.90 (q, J=8.8 Hz, 2H), 3.79 (dd, J=5.6, 3.5 Hz, 2H),3.71-3.61 (m, 6H).

Step 2: Synthesis of 2-[2-(2,2,2-trifluoroethoxy)ethoxy]

To a solution of 2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxymethylbenzene(0.5 g, 1.80 mmol) in CH₃OH (10 mL) was added Pd/C (436.45 mg). Thismixture was then stirred under H₂ atmosphere at room temperature for 20h, filtered and the filtrate concentrated and purified via silica gelchromatography (DCM: CH₃OH=50: 1) to obtain2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethanol (0.30 g, 89% yield) ascolorless oil. ¹H NMR (400 MHz, CDCl₃) δ 3.91 (q, J=8.7 Hz, 2H), 3.80(dd, J=5.6, 3.4 Hz, 2H), 3.75 (d, J=4.0 Hz, 2H), 3.70 (dd, J=5.5, 3.5Hz, 2H), 3.62 (dd, J=5.2, 3.9 Hz, 2H), 2.23 (t, J=5.7 Hz, 1H (OH)). ¹⁹FNMR (376 MHz, CDCl₃, (trifluoromethyl)benzene as standard) 8-74.33 (t,J=8.8 Hz).

Step 3: Synthesis of(21E,23E,25E,26E,35R,36S,37R,38R,40S,42S,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-35,36,37,38,48,49-hexamethyl-44-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(48),26(49)-tetraene-50,51,52,53,54-pentone(I-86)

A solution of everolimus (0.5 g, 0.52 mmol) in THF (5 mL) was degassed,p-toluenesulfonic acid (0.45 g, 2.61 mmol) was added at 0° C. followedby 2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethanol (0.98 g, 5.22 mmol). Theresulting mixture was stirred at 0° C. for 0.5 h under N₂, then at 23°C. for 6 h, poured into sat.NaHCO₃(40 mL) and extracted with EtOAc (30mL). The organic layer was washed with water (30 mL×2), brine (40 mL),dried over anhydrous Na₂SO₄, filtered, and concentrated under reducedpressure. The residue was purified by reverse phase chromatography (C18,CH₃CN:H₂O from 0% to 70% yield) to provide(21E,23E,25E,26E,35R,36S,37R,38R,40S,42S,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-35,36,37,38,48,49-hexamethyl-44-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(48),26(49)-tetraene-50,51,52,53,54-pentone(I-86: 0.08 g, 14% yield) as a white solid. ESI-MS (EI⁺, m/z): 1136.5[M+Na]⁺. ¹H NMR (400 MHz, CDCl₃): δ 6.44-5.88 (m, 4H), 5.73-5.06 (m,4H), 4.52-4.32 (m, 1H), 4.22-4.12 (m, 1H), 3.91-3.81 (m, 2H), 3.71-3.51(m, 6H), 3.42-3.21 (m, 16H), 3.13-2.98 (m, 4H), 2.63-2.42 (m, 4H),2.32-2.14 (m, 2H), 2.05-1.93 (m, 3H), 1.86-1.55 (m, 16H), 1.44-1.35 (m,4H), 1.24-1.15 (m, 5H), 1.06-0.78 (m, 17H), 0.65-0.51 (m, 1H).

Step 4: Synthesis of(21E,23E,25E,26E,35R,36S,37R,38R,40S,42S,44S,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-35,36,37,38,48,49-hexamethyl-44-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(48),26(49)-tetraene-50,51,52,53,54-pentone(I-85)

100 mg of(21E,23E,25E,26E,35R,36S,37R,38R,40S,42S,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-35,36,37,38,48,49-hexamethyl-44-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(48),26(49)-tetraene-50,51,52,53,54-pentonewas purified via prep chiral HPLC which provided(21E,23E,25E,26E,35R,36S,37R,38R,40S,42S,44S,45S,46R,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-35,36,37,38,48,49-hexamethyl-44-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(48),26(49)-tetraene-50,51,52,53,54-pentone(I-85: 14.3 mg, 14.3% yield) as a white solid.

Chiral Separation Method:

Column: CHIRALPAK IC

Column size: 5.0 cm I.D.×25 cm L

Solution concentration: 2.4 mg/ml

Injection: 5 ml

Mobile phase: Hexane/EtOH=70/30(V/V)

Flow rate: 30 ml/min

Wave length: UV 254 nm

Temperature: 35° C.

I-85: ESI-MS (EI⁺, m/z): 1136.4 [M+Na]⁺. ¹H NMR (400 MHz, CDCl3) δ6.42-6.06 (m, 3H), 5.92 (dd, J=30.3, 10.3 Hz, 1H), 5.56-5.06 (m, 5H),4.74 (s, 1H), 4.18 (d, J=5.7 Hz, 1H), 3.94-3.83 (m, 2H), 3.82-3.51 (m,12H), 3.49-3.25 (m, 11H), 3.22-3.03 (m, 2H), 2.72 (dd, J=16.6, 5.5 Hz,2H), 2.57 (dd, J=17.0, 6.5 Hz, 1H), 2.34 (d, J=12.4 Hz, 2H), 2.25-2.18(m, 1H), 2.13-1.85 (m, 5H), 1.69 (dd, J=35.2, 8.9 Hz, 10H), 1.47 (dd,J=20.5, 13.6 Hz, 5H), 1.26 (s, 7H), 1.15-0.81 (m, 18H), 0.71 (dd,J=23.9, 12.0 Hz, 1H).

Example 38: Synthesis of(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone(I-91) and(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,42S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone(I-85) and(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,42R,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethanol

2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethanol was same as Example 37.

Step 2: Synthesis of(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone(I-91)

A solution of rapamycin (0.5 g, 0.547 mmol) in DCM (20 mL) was degassedat −40° C., and trifluoroacetic acid (1.67 mL) was added. After stirringfor 10 min. 2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethanol (0.2 g, 1.09mmol) was added. The mixture was stirred at −40° C. for a further 40min. then poured into ice cold NaHCO₃ (aq. 60 mL), washed with water (20mL), brine (20 mL), dried over MgSO₄, filtered and concentrated. Theresidue was purified by reverse phase chromatography (C18,CH₃CN:H₂O=65:35) to provide(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone(I-91: 85 mg, 15% yield) as a white solid. ESI-MS (EI⁺, m/z): 1092.4[M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ 6.45-5.76 (m, 4H), 5.40 (ddd, J=24.9,15.2, 8.0 Hz, 2H), 5.25-4.99 (m, 2H), 4.57-4.01 (m, 3H), 3.98-3.45 (m,7H), 3.43-2.99 (m, 11H), 2.95-2.37 (m, 6H), 2.26 (d, J=13.9 Hz, 2H),2.08-1.76 (m, 6H), 1.75-1.52 (m, 14H) 1.48-1.10 (m, 10H), 1.07-0.74 (m,18H), 0.60 (dd, J=23.5, 12.0 Hz, 1H).

Step 3: Synthesis of(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,42R,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone(I-126)

159 mg of(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentonewas purified via prep chiral HPLC to provide(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,42S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone(I-125: 48.5 mg, 30% yield) and(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,42R,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-[2-(2,2,2-trifluoroethoxy)ethoxy]ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone(I-126:43 mg, 27% yield) as a white solid.

Chiral Separation Method:

Column: CHIRALPAK IC

Column size: 5.0 cm I.D.×25 cm L

Solution concentration: 3.0 mg/ml

Injection: 3 ml

Mobile phase: Hexane/EtOH=70/30(V/V)

Flow rate: 30 ml/min

Wave length: UV 254 nm

Temperature: 35° C.

I-125: ESI-MS (EI⁺, m/z): 1092.4 [M+Na]. ¹H NMR (400 MHz, CDCl₃) δ6.36-6.13 (m, 2H), 6.06 (dd, J=15.0, 10.0 Hz, 1H), 5.85 (dd, J=29.4,10.8 Hz, 1H), 5.49-5.37 (m, 1H), 5.33 (d, J=10.0 Hz, 1H), 5.20 (d, J=4.6Hz, 1H), 5.08 (t, J=11.7 Hz, 1H), 4.68 (s, 1H), 4.11 (d, J=5.8 Hz, 1H),3.94-3.64 (m, 7H), 3.63-3.46 (m, 5H), 3.42-3.18 (m, 12H), 2.93-2.79 (m,2H), 2.71-2.43 (m, 4H), 2.27 (d, J=11.8 Hz, 2H), 2.08-1.85 (m, 6H),1.83-1.61 (m, 11H), 1.48-1.21 (m, 8H), 1.08-0.74 (m, 18H), 0.65-0.54 (m,1H).

I-126: ESI-MS (EI⁺, m/z): 1092.4 [M+Na]⁺. ¹H NMR (400 MHz, CDCl3) δ6.41-6.07 (m, 3H), 6.00-5.81 (m, 1H), 5.56-5.05 (m, 4H), 4.75 (s, 1H),4.18 (d, J=5.8 Hz, 1H), 3.98-3.52 (m, 11H), 3.50-3.22 (m, 12H), 2.95 (d,J=8.6 Hz, 1H), 2.77-2.50 (m, 4H), 2.38-2.16 (m, 2H), 2.12-1.83 (m, 5H),1.69 (dd, J=39.3, 11.0 Hz, 12H), 1.49-1.17 (m, 11H), 1.15-0.80 (m, 18H),0.74-0.60 (m, 1H).

Example 39: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2-(trifluoromethoxy)ethoxy]ethoxy]-66,67-dioxa-57-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-88) and(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43R,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2-(trifluoromethoxy)ethoxy]ethoxy]-66,67-dioxa-57-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-82) and(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2-(trifluoromethoxy)ethoxy]ethoxy]-66,67-dioxa-57-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-83)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis ofO-[2-(2-benzyloxyethoxy)ethyl] Methylsulfanylmethanethioate

A two-necked 1000 mL round-bottom flask equipped with a magnetic stirbar was charged with 2-(2-benzyloxyethoxy)ethanol (12 g, 61.2 mmol) andbenzyl(triethyl)ammonium chloride (1.0 g, 4.87 mmol). A 50% aqueoussolution of sodium hydroxide (141 mL) was added via a dropping funnel.After the mixture was stirred for 10 min, CS₂ (141 mL, 2.34 mol) wasadded dropwise, followed by iodomethane (22.0 g, 154 mmol). The mixturewas stirred for 3 h at room temperature. Water (100 mL) was added. Theorganic layer was removed, and the aqueous phase was extracted withCH₂C2 (100 mL×3). The combined organic layers were washed with brine(2×100 mL), dried over MgSO₄, filtered and concentrated. The residue waspurified via silica gel chromatography (EtOAc:PE=1:3) to obtain0-[2-(2-benzyloxyethoxy)ethyl] methylsulfanylmethanethioate (16.8 g, 95%yield) as a yellow oil. ESI-MS (EI⁺, m/z): 308.9 [M+Na]⁺. ¹H NMR (500MHz, DMSO-d₆) δ 7.32 (dt, J=18.3, 6.8 Hz, 5H), 4.74-4.62 (m, 2H), 4.50(s, 2H), 3.85-3.72 (m, 2H), 3.60 (ddd, J=8.7, 6.2, 3.6 Hz, 4H), 2.56 (s,3H).

Step 2: Synthesis of 2-[2-(trifluoromethoxy)ethoxy]ethoxymethylbenzene

To a suspension of 1,3-dibromo-5,5-dimethyl-imidazolidine-2,4-dione(29.95 g, 104.75 mmol) in DCM (150 mL) was added (HF)₉/Py pyridiniumpoly (hydrogen fluoride) (49.36 mL, 209.49 mmol,) andO-[2-(2-benzyloxyethoxy)ethyl] methylsulfanylmethanethioate (10 g, 34.92mmol) at −78° C., and the mixture was stirred at −50° C. for 2 h. Themixture was poured into an aqueous solution of NaHCO₃ and NaHSO₃, driedover anhydrous Na₂SO₄, filtered, and concentrated, then purified viasilica gel chromatography (PE:EtOAc=25:1) to provide the crude product.Then the crude was further purified via silica gel chromatography(PE:EtOAc=100:1 to 50:1 to 40:1) to obtain2-[2-(trifluoromethoxy)ethoxy]ethoxymethylbenzene (3.6 g, 39% yield) ascolorless liquid. ESI-MS (EI⁺, m/z): 287.1 [M+Na]⁺. ¹H NMR (400 MHz,CDCl₃) (7.37-7.22 (m, 5H), 4.57 (s, 2H), 4.10 (t, J=4.8 Hz, 2H), 3.75(t, J=4.8 Hz, 2H), 3.72-3.62 (m, 4H).

Step 3: Synthesis of 2-[2-(trifluoromethoxy)ethoxy]ethanol

To a solution of 2-[2-(trifluoromethoxy)ethoxy]ethoxymethylbenzene (3.4g, 12.87 mmol) in CH₃OH (60 mL) was added Pd/C (3.13 g). This mixturewas then stirred under H₂ atmosphere at room temperature for 20 h,filtered, concentrated then purified by silica gel chromatography (DCM:CH₃OH=50: 1) to provide 2-[2-(trifluoromethoxy)ethoxy]ethanol (1.87 g,83% yield) as colorless liquid. ¹H NMR (400 MHz, CDCl₃) δ 4.11-4.03 (m,2H), 3.69 (dt, J=4.4, 2.3 Hz, 4H), 3.61-3.54 (m, 2H), 2.75 (t, J=5.9 Hz,1H). ¹⁹F NMR (376 MHz, CDCl3) (5-61.11 (s).

Step 4: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2-(trifluoromethoxy)ethoxy]ethoxy]-66,67-dioxa-57-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-88)

To a degassed solution of everolimus (0.5 g, 0.52 mmol) in THF (30 mL)was added p-toluenesulfonic acid (0.45 g, 2.61 mmol) at 0° C. followedby 2-[2-(trifluoromethoxy)ethoxy]ethanol (0.91 g, 5.22 mmol). Theresulting mixture was stirred at 0° C. for 0.5 h under N₂, then at 23°C. for 6 h. The reaction was poured into sat.NaHCO₃(40 mL) and extractedwith EtOAc (30 mL). The organic layer was washed with water (30 mL×2),brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentratedunder reduced pressure at room temperature. The residue was purified byreverse phase chromatography (C18, CH₃CN: H₂O=0% to 70% yield) toprovide(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2-(trifluoromethoxy)ethoxy]ethoxy]-66,67-dioxa-57-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-88: 141 mg, 24% yield) as a white solid. ESI-MS (EI⁺, m/z): 1122.3[M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) (6.39-5.88 (m, 4H), 5.73-5.05 (m, 5H),4.52-3.83 (m, 5H), 3.70-3.50 (m, 6H), 3.43-3.21 (m, 8H), 3.12-2.93 (m,4H), 2.80-2.44 (m, 4H), 2.31-2.16 (m, 4H), 2.05-1.59 (m, 20H), 1.43-1.34(m, 4H), 1.21-1.09 (m, 6H), 1.01-0.78 (m, 17H), 0.62-0.51 (m, 1H).

Step 5: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2-(trifluoromethoxy)ethoxy]ethoxy]-66,67-dioxa-57-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-83) and(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43R,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2-(trifluoromethoxy)ethoxy]ethoxy]-66,67-dioxa-57-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-82)

130 mg of(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2-(trifluoromethoxy)ethoxy]ethoxy]-66,67-dioxa-57-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentonewas purified via prep chiral HPLC to provide(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43R,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2-(trifluoromethoxy)ethoxy]ethoxy]-66,67-dioxa-57-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-82: 19 mg, 14.6% yield) and(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2-(trifluoromethoxy)ethoxy]ethoxy]-66,67-dioxa-57-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-83: 12 mg, 9.2% yield), both as white solids.

Chiral Analysis Method:

Column: CHIRALPAK IC(IC00CE-OL002)

Column size: 0.46 cm I.D.×25 cm L

Injection: 40.0 ul

Mobile phase: Hexane/EtOH=60/40(V/V)

Flow rate: 1.0 ml/min

Wave length: UV 254 nm

Temperature: 35° C.

HPLC equipment: Shimadzu LC-20AT CP-HPLC-07

I-82: ESI-MS (EI⁺, m/z): 1122.4 [M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ6.43-5.83 (m, 4H), 5.57-5.13 (m, 5H), 4.31-4.04 (m, 3H), 3.91-3.53 (m,11H), 3.49-3.00 (m, 19H), 2.76-2.52 (m, 3H), 2.25 (dd, J=34.2, 26.6 Hz,3H), 2.12-1.96 (m, 5H), 1.75 (dd, J=35.2, 24.7 Hz, 8H), 1.52-1.34 (m,8H), 1.15-0.79 (m, 18H), 0.72 (d, J=12.1 Hz, 1H).

I-83: ESI-MS (EI⁺, m/z): 1122.4 [M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ6.54-6.41 (m, 1H), 6.18 (tdd, J=29.7, 22.4, 12.7 Hz, 3H), 5.82-5.68 (m,1H), 5.46-5.30 (m, 2H), 5.19 (dd, J=25.5, 20.6 Hz, 2H), 4.62-4.40 (m,1H), 4.21 (d, J=18.4 Hz, 1H), 3.94 (dd, J=34.8, 4.5 Hz, 1H), 3.83-3.62(m, 4H), 3.59 (d, J=3.3 Hz, 1H), 3.50-2.95 (m, 13H), 2.62 (dt, J=55.5,38.6 Hz, 2H), 2.42-2.17 (m, 3H), 2.16-1.57 (m, 24H), 1.54-1.27 (m, 10H),1.12-0.80 (m, 18H), 0.71-0.62 (m, 1H).

Example 40: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-87) and(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43R,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-79) and(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-80)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxymethylbenzene

Under a nitrogen atmosphere, silver trifluoromethane sulfonate (19.64 g,76.44 mmol), lithium trifluoromethane sulfonate (3.97 g, 25.48 mmol),1-(chloromethyl)-4-fluoro-1,4-diazoniabicyclo[2.2.2]octanebis(tetrafluoroborate) (18.05 g, 50.96 mmol) and potassium fluoride(5.92 g, 101.92 mmol) were mixed. 2-(2-benzyloxyethoxy)ethanol (5 g,25.48 mmol), trimethyl(1,1,2,2,2-pentafluoroethyl)silane (14.69 g, 76.44mmol), EtOAc (20 mL), trifluoromethylbenzene (20 mL), and2-fluoropyridine (7.42 g, 76.44 mmol) were then added under N₂atmosphere in this order. The reaction mixture was stirred under N₂atmosphere for 60 h at 30° C. then filtered through a plug of silica(eluting with EtOAc). The filtrate was collected and concentrated. Theresidue was purified via silica gel chromatography (PE:EtOAc=25:1) toobtain 2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxymethylbenzene (2.5g, 31% yield) as a light yellow liquid. ¹H NMR (400 MHz, CDCl₃) δ7.36-7.24 (m, 5H), 4.56 (s, 2H), 4.17-4.11 (m, 2H), 3.76-3.72 (m, 2H),3.71-3.65 (m, 2H), 3.65-3.60 (m, 2H). ¹⁹F NMR (376 MHz, CDCl₃) δ −90.77(s), −86.02 (s).

Step 2: Synthesis of 2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethanol

To a solution of2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxymethylbenzene (0.757 g,2.41 mmol) in CH₃OH (10 mL) was added Pd/C (0.58 g). The mixture wasstirred under H₂ atmosphere at room temperature for 20 h then filteredand concentrated. The residue was purified by silica gel chromatography(DCM:CH₃OH=50:1) to obtain2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethanol (0.5 g, 93% yield) as acolorless liquid. ¹H NMR (400 MHz, CDCl₃) δ 4.13-4.06 (m, 2H), 3.69 (dd,J=5.4, 4.1 Hz, 4H), 3.54 (dd, J=5.2, 3.9 Hz, 2H), 2.47 (s, 1H). ¹⁹F NMR(376 MHz, CDCl₃) δ −86.21 (d, J=1.2 Hz), −90.98 (d, J=1.2 Hz).

Step 3: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-87)

To a solution of everolimus (0.5 g, 0.52 mmol) in THF (10 mL) was addedp-toluenesulfonic acid (0.45 g, 2.61 mmol) at 0° C., followed by2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethanol (0.58 g, 2.61 mmol).The resulting mixture was stirred at 0° C. for 0.5 h under N₂, then at22° C. for 6 h, poured into sat. NaHCO₃(40 mL) and extracted with EtOAc(30 mL). The organic layer was washed with water (30 mL×2), brine (20mL), dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure at room temperature. The residue was purified byreverse phase chromatography (C18, CH₃CN: H₂O=0% to 70% yield) toprovide(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-87:40 mg, 7% yield) as a white solid. ESI-MS (EI⁺, m/z): 1172.3[M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ 6.45-5.95 (m, 4H), 5.79-5.13 (m, 5H),4.59-4.17 (m, 3H), 3.98-3.59 (m, 8H), 3.50-3.28 (m, 1OH), 3.20-3.00 (m,5H), 2.89-2.49 (m, 4H), 2.37-2.22 (m, 4H), 2.10-1.61 (m, 20H), 1.51-1.23(m, 8H), 1.06-0.85 (m, 16H), 0.71-0.63 (m, 1H).

Step 4: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-80) and(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43R,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-79)

95 mg of(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentonewas sent for chiral separation to obtain(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-80: 7.2 mg, 7.5% yield) and(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43R,44S,45R,46R,55R)-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-43-[2-[2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]ethoxy]-67,68-dioxa-58-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-79: 5.1 mg, 5.3% yield) as a white solid.

Chiral Separation Method:

Column: CHIRALPAK IC

Column size: 5.0 cm I.D.×25 cm L

Solution concentration: 0.79 mg/ml

Injection: 5 ml

Mobile phase: Hexane/EtOH=70/30(V/V)

Flow rate: 30 ml/min

Wave length: UV 254 nm

Temperature: 35° C.

I-80: ESI-MS (EI⁺, m/z): 1150.3 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ6.42-5.80 (m, 4H), 5.58-5.05 (m, 5H), 4.74 (s, 1H), 4.24-4.08 (m, 3H),3.92-3.51 (m, 10H), 3.48-3.24 (m, 12H), 3.13 (ddd, J=24.2, 17.1, 11.3Hz, 3H), 2.64 (ddd, J=23.4, 16.8, 6.0 Hz, 3H), 2.42-2.15 (m, 3H),2.14-1.88 (m, 6H), 1.84-1.64 (m, 14H), 1.54-1.39 (m, 5H), 1.17-0.81 (m,18H), 0.71 (dd, J=23.8, 12.1 Hz, 1H).

I-79: ESI-MS (EI⁺, m/z): 1172.3 [M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ6.42-5.90 (m, 4H), 5.54-5.03 (m, 6H), 4.31-3.96 (m, 5H), 3.88-3.01 (m,22H), 2.94-2.37 (m, 5H), 2.39-1.94 (m, 7H), 1.68 (dd, J=28.2, 19.2 Hz,8H), 1.52-1.31 (m, 6H), 1.14-1.00 (m, 6H), 0.97-0.62 (m, 19H).

Example 41: Synthesis of(21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-(2-hydroxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone(I-76) and(21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,45S,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-(2-hydroxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone(I-66) and(21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,45S,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-(2-hydroxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone(I-67)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of2-[tert-butyl(diphenyl)silyl]oxyethyl Trifluoromethanesulfonate

A solution of 2-[tert-butyl(diphenyl)silyl]oxyethanol (4.3 g, 14.31mmol) and DIPEA (2.77 g, 21.47 mmol) in DCM (40 mL) was cooled to 0° C.under N₂ and trifluoromethanesulfonic anhydride (4.44 g, 15.74 mmol)added dropwise. The mixture was stirred at 0° C. for 2 h then dilutedwith DCM (50 mL), washed with sat. NaHCO₃ (50 mL), water (50 mL×3), andbrine (50 mL). The organic layer was then dried over MgSO₄, filtered,and concentrated under vacuum to afford2-[tert-butyl(diphenyl)silyl]oxyethyl trifluoromethanesulfonate (6.19 g,99% yield) as a brown oil. This was used in the next step without anyfurther purification. ¹H NMR (400 MHz, CDCl₃) δ 7.66 (dd, J=7.9, 1.5 Hz,4H), 7.45-7.38 (m, 6H), 4.56 (t, J=4.4 Hz, 2H), 3.91 (t, J=4.4 Hz, 2H).

Step 2: Synthesis of(35E,37E,39E,40E,48R,49S,50R,51R,53S,55S,57S,58S,59R,60R,69R)-58-[(1R)-2-[(1S,3R,4R)-4-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-59,69-dihydroxy-57,60-dimethoxy-48,49,50,51,61,62-hexamethyl-79,80-dioxa-71-azatricyclohexatriaconta-35,37,39(61),40(62)-tetraene-63,64,65,66,67-pentone

A solution of everolimus (1.5 g, 1.57 mmol),2-[tert-butyl(diphenyl)silyl]oxyethyl trifluoromethanesulfonate andDIPEA (3.27 mL, 18.78 mmol) in toluene (20 mL) was stirred at 45° C. for18 h. The mixture was then poured into ice cold sat. NaHCO₃(50 mL),washed with ice-water twice (60 mL), brine (50 mL), dried over anhydrousNa₂SO₄, filtered, and concentrated. The mixture was purified via silicagel chromatography (PE:EtOAc=5:1 to 2:1, then PE:acetone=4:1) to obtain(35E,37E,39E,40E,48R,49S,50R,51R,53S,55S,57S,58S,59R,60R,69R)-58-[(1R)-2-[(1S,3R,4R)-4-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-59,69-dihydroxy-57,60-dimethoxy-48,49,50,51,61,62-hexamethyl-79,80-dioxa-71-azatricyclohexatriaconta-35,37,39(61),40(62)-tetraene-63,64,65,66,67-pentone(1.15 g, 59% yield) as a brown solid. ESI-MS (EI⁺, m/z): 1263.4 [M+Na]⁺.¹H NMR (400 MHz, CDCl₃) δ 7.62-7.60 (m, 4H), 7.35-7.28 (m, 6H),6.35-5.80 (m, 4H), 5.51-5.09 (m, 4H), 4.75 (s, 1H), 4.37-4.02 (m, 2H),3.87-3.49 (m, 11H), 3.37-3.26 (m, 8H), 3.10-2.96 (m, 5H), 2.76-2.48 (m,3H), 2.28-2.21 (m, 2H), 1.98-1.90 (m, 3H), 1.67-1.39 (m, 18H), 1.26-1.08(m, 7H), 1.04-0.76 (m, 26H), 0.64 (q, J=11.2 Hz, 1H).

Step 3: Synthesis of(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,42S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-42,45-dimethoxy-33,34,35,36,46,47-hexamethyl-64,65-dioxa-55-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraene-48,49,50,51,52-pentone

(35E,37E,39E,40E,48R,49S,50R,51R,53S,55S,57S,58S,59R,60R,69R)-58-[(1R)-2-[(1S,3R,4R)-4-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-59,69-dihydroxy-57,60-dimethoxy-48,49,50,51,61,62-hexamethyl-79,80-dioxa-71-azatricyclohexatriaconta-35,37,39(61),40(62)-tetraene-63,64,65,66,67-pentone(1.15 g, 0.93 mmol) was dissolved in THF (10 mL). Pyridiniumhydrofluoride (0.437 mL, 1.85 mmol,) was added and the mixture stirredfor 3 h at room temperature. The mixture was diluted with EtOAc (30 mL),washed with sat. NaHCO₃ (aq., 40 mL x 2) until pH 10, then washed withwater until neutral, brine (40 mL), dried over anhydrous Na₂SO₄,filtered, and concentrated. The residue was purified via silica gelchromatography (PE:acetone=4:1 to 2:1) to obtain(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,42S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-42,45-dimethoxy-33,34,35,36,46,47-hexamethyl-64,65-dioxa-55-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraene-48,49,50,51,52-pentone(680 mg, 73% yield) as a white solid. ESI-MS (EI⁺, m/z): 1024.3 [M+Na]⁺.¹H NMR (400 MHz, CDCl₃) 6.35-5.80 (m, 4H), 5.51-5.09 (m, 4H), 4.75 (s,1H), 4.37-4.02 (m, 2H), 3.87-3.49 (m, 11H), 3.37-3.26 (m, 8H), 3.10-2.96(m, 5H), 2.76-2.48 (m, 3H), 2.28-2.21 (m, 2H), 1.98-1.90 (m, 3H),1.67-1.39 (m, 18H), 1.26-1.08 (m, 7H), 1.04-0.76 (m, 17H), 0.64 (q,J=11.2 Hz, 1H).

Step 4: Synthesis of(21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-(2-hydroxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone(I-76)

To a solution of(22E,24E,26E,27E,33R,34S,35R,36R,38S,40S,42S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-42,45-dimethoxy-33,34,35,36,46,47-hexamethyl-64,65-dioxa-55-azatricyclohexatriaconta-22,24,26(46),27(47)-tetraene-48,49,50,51,52-pentone(0.65 g, 0.65 mmol) in THF (6 mL) under N2 at 0° C. was added p-TsOH(0.56 g, 3.24 mmol) followed by 2-(2-hydroxyethoxy)ethanol (1.38 g,12.97 mmol). The resulting mixture was stirred at 0° C. for 10 min, thenat 20° C. for 2 h. The mixture was poured into sat.NaHCO₃(40 mL) andextracted with EtOAc (30 mL). The organic layer was washed with water(30 mL×2), brine (40 mL), dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure at room temperature. The residue waspurified via silica gel chromatography (PE:EtOAc=50% to 100% EtOAc, thento DCM:CH₃OH=95:5 to 90:10) then purified by reverse-phasechromatography (C18, CH₃CN:H₂O=50: 50) to provide(21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-(2-hydroxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone(I-76: 140 mg, 20% yield) as a white solid. ESI-MS (EI⁺, m/z): 1098.5[M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ 6.31-5.80 (m, 4H), 5.46-4.74 (m, 5H),4.21-4.11 (m, 2H), 3.68-3.56 (m, 18H), 3.4-3.14 (m, 12H), 3.03-2.97 (m,2H), 2.68-2.39 (m, 5H), 2.28-2.25 (d, 2H), 1.73-1.55 (m, 18H), 1.40-1.14(m, 8H), 1.03-0.79 (m, 16H), 0.64 (q, J=11.2 Hz, 1H).

Step 5: Synthesis of(21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,45S,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-(2-hydroxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone(I-67) and(21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,45S,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-(2-hydroxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone(I-66)

140 mg of(21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-(2-hydroxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentonepurified via prep chiral HPLC and the resulting epimers repurified viasilica gel chromatography (hexane:DCM:EtOAc:MeOH=3:3:1:1) to provide(21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,45S,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-(2-hydroxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone(I-67: 25.3 mg, 18.1% yield) and(21E,23E,25E,26E,36R,37S,38R,39R,41S,43S,45S,46S,47R,48R,57R)-47,57-dihydroxy-45-[2-(2-hydroxyethoxy)ethoxy]-46-[(1R)-2-[(1S,3R,4R)-4-[2-(2-hydroxyethoxy)ethoxy]-3-methoxy-cyclohexyl]-1-methyl-ethyl]-48-methoxy-36,37,38,39,49,50-hexamethyl-68,69-dioxa-58-azatricyclohexatriaconta-21,23,25(49),26(50)-tetraene-51,52,53,54,55-pentone(I-66: 34 mg, 24.3% yield), both as white solids.

Chiral Analysis Method:

Column: CHIRALPAK IC(IC00CD-OL002)

Column size: 0.46 cm I.D.×25 cm L

Injection: 100.0 ul

Mobile phase: Hexane/EtOH=60/40(V/V)

Flow rate: 1.0 ml/min

Wave length: UV 254 nm

Temperature: 35° C.

HPLC equipment: Shimadzu LC-20AD CP-HPLC-08

I-67: ESI-MS (EI⁺, m/z): 1098.4 [M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ6.49-6.19 (m, 2H), 6.13 (dd, J=15.0, 9.8 Hz, 1H), 5.94 (dd, J=24.3, 10.5Hz, 1H), 5.58-5.45 (m, 1H), 5.41 (d, J=9.8 Hz, 1H), 5.37-5.24 (m, 1H),5.13 (t, J=10.8 Hz, 1H), 4.80 (s, 1H), 4.19 (t, J=8.7 Hz, 1H), 3.92-3.52(m, 16H), 3.51-3.25 (m, 11H), 3.25-2.99 (m, 3H), 2.72 (dd, J=16.9, 5.6Hz, 2H), 2.57 (dd, J=16.8, 6.3 Hz, 1H), 2.39-2.18 (m, 2H), 1.95 (ddd,J=29.6, 22.0, 10.2 Hz, 6H), 1.83-1.40 (m, 15H), 1.37-1.16 (m, 8H),1.15-0.82 (m, 18H), 0.71 (dd, J=24.0, 12.0 Hz, 1H).

I-66: ESI-MS (EI⁺, m/z): 1098.4 [M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ6.45-5.92 (m, 4H), 5.52-4.75 (m, 5H), 4.31-3.92 (m, 3H), 3.88-3.54 (m,16H), 3.51-3.13 (m, 13H), 3.07 (s, 2H), 2.87-2.42 (m, 4H), 2.38-1.55 (m,12H), 1.51-1.29 (m, 15H), 1.13-0.72 (m, 18H), 0.69-0.58 (m, 1H).

Example 42: Synthesis of(22E,24E,26E,27E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-43-hexoxy-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-65,66-dioxa-56-azatricyclohexatriaconta-22,24,26(47),27(48)-tetraene-49,50,51,52,53-pentone(I-103)

Synthetic Scheme:

Procedures and Characterization:

Step 1: Synthesis of(22E,24E,26E,27E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-43-hexoxy-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-65,66-dioxa-56-azatricyclohexatriaconta-22,24,26(47),27(48)-tetraene-49,50,51,52,53-pentone(I-103)

To a solution of rapamycin (0.5 g, 0.547 mmol) and hexan-1-ol (56 mg,0.547 mmol) in THF (10 mL) was added 4-methylbenzenesulfonic acidhydrate (0.52 g, 2.73 mmol) slowly. The resulting solution was stirredat 20° C. for 2h under N₂, then the mixture was poured into ice cold aq.NaHCO₃ and extracted with EtOAc (30 mL). The organic layer was dried,filtered and concentrated. The residue was then purified via reversephase chromatography (C18, CH₃CN:H₂O=78:22) to afford(22E,24E,26E,27E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,55R)-43-hexoxy-45,55-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-65,66-dioxa-56-azatricyclohexatriaconta-22,24,26(47),27(48)-tetraene-49,50,51,52,53-pentone(I-103: 72 mg, 13% yield) as a white solid. ESI-MS (EI⁺, m/z): 1006.0[M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ 6.44-5.85 (m, 4H), 5.60-5.07 (m, 4H),4.77 (s, 1H), 4.29-3.98 (m, 2H), 3.76-3.67 (m, 1H), 3.46-3.28 (m, 10H),3.23-3.03 (m, 2H), 3.02-2.77 (m, 2H), 2.69 (m, 3H), 2.36 (d, J=32.6 Hz,2H), 2.17-1.90 (m, 4H), 1.66-1.44 (m, 22H), 1.35-1.21 (m, 11H),1.17-0.84 (m, 22H), 0.72-0.65 (m, 1H).

Example 43: Synthesis of(21E,23E,25E,26E,44R,45S,46R,47R,49S,51S,54S,55R,56R,65R)-55,65-dihydroxy-53-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-54-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-56-methoxy-44,45,46,47,57,58-hexamethyl-76,77-dioxa-66-azatricyclohexatriaconta-21,23,25(57),26(58)-tetraene-59,60,61,62,63-pentone(I-99)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of(21E,23E,25E,26E,44R,45S,46R,47R,49S,51S,54S,55R,56R,65R)-55,65-dihydroxy-53-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-54-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-56-methoxy-44,45,46,47,57,58-hexamethyl-76,77-dioxa-66-azatricyclohexatriaconta-21,23,25(57),26(58)-tetraene-59,60,61,62,63-pentone(I-99)

To a solution of everolimus (0.5 g, 0.52 mmol) and2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol(3.41 g, 10.44 mmol) in THF (15 mL) was added 4-methylbenzenesulfonicacid hydrate (0.52 g, 2.73 mmol) slowly. The resulting mixture wasstirred at 22° C. for 18 h under N₂ then quenched with aq. NaHCO₃ (30mL) and extracted with EtOAc (60 mL×3). The combined organic layers werewashed with water (50 mL), brine (50 mL), dried over anhydrous Na₂SO₄,filtered, and concentrated under vacuum. The residue was purified byreverse phase chromatography (C18, CH₃CN:H₂O=70:30) to afford(21E,23E,25E,26E,44R,45S,46R,47R,49S,51S,54S,55R,56R,65R)-55,65-dihydroxy-53-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-54-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-56-methoxy-44,45,46,47,57,58-hexamethyl-76,77-dioxa-66-azatricyclohexatriaconta-21,23,25(57),26(58)-tetraene-59,60,61,62,63-pentone(I-99: 93 mg, 14% yield) as a white solid. ESI-MS (EI⁺, m/z): 1274.9[M+Na]⁺. ¹H NMR (500 MHz, CDCl3) δ 6.42-5.82 (m, 4H), 5.64-5.06 (m, 4H),4.81 (s, 1H), 4.30-4.08 (m, 1H), 3.81-3.53 (m, 35H), 3.46-3.26 (m, 12H),3.22-3.05 (m, 4H), 2.76-2.65 (m, 2H), 2.39-2.22 (m, 2H), 2.14-1.97 (m,3H), 1.75-1.55 (m, 13H), 1.52-1.39 (m, 4H), 1.30-1.13 (m, 6H), 1.08-0.82(m, 17H), 0.76-0.65 (m, 1H).

Example 44: Synthesis of(21E,23E,25E,26E,46R,47S,48R,49R,51S,53S,56S,57R,58R,67R)-57,67-dihydroxy-55-[2-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-56-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-58-methoxy-46,47,48,49,59,60-hexamethyl-78,79-dioxa-68-azatricyclohexatriaconta-21,23,25(59),26(60)-tetraene-61,62,63,64,65-pentone(I-97)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of(21E,23E,25E,26E,46R,47S,48R,49R,51S,53S,56S,57R,58R,67R)-57,67-dihydroxy-55-[2-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-56-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-58-methoxy-46,47,48,49,59,60-hexamethyl-78,79-dioxa-68-azatricyclohexatriaconta-21,23,25(59),26(60)-tetraene-61,62,63,64,65-pentone(I-97)

To a solution of rapamycin (0.5 g, 0.547 mmol) in THF (15 mL) was added4-methylbenzenesulfonic acid (0.47 g, 2.73 mmol) and nonaethylene glycol(2.27 g, 5.47 mmol) at 10° C. The reaction was stirred at 30° C. for 18h under N₂ then quenched with aq. NaHCO₃ and extracted with EtOAc (60mL×3). The combined organic layers were washed with water (50 mL), brine(50 mL), dried over anhydrous Na₂SO₄, filtered and concentrated. Theresidue was purified by reverse phase chromatography (C18,CH₃CN:H₂O=63:37) to provide(21E,23E,25E,26E,46R,47S,48R,49R,51S,53S,56S,57R,58R,67R)-57,67-dihydroxy-55-[2-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-56-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-58-methoxy-46,47,48,49,59,60-hexamethyl-78,79-dioxa-68-azatricyclohexatriaconta-21,23,25(59),26(60)-tetraene-61,62,63,64,65-pentone(I-97: 111 mg, 16% yield) as a white solid. ESI-MS (EI⁺, m/z): 1318.9[M+Na]⁺. ¹H NMR (500 MHz, CDCl3) δ 6.50-5.79 (m, 4H), 5.64-5.06 (m, 4H),4.89 (d, J=53.5 Hz, 1H), 4.51-3.94 (m, 2H), 3.75-3.18 (m, 46H),3.02-2.86 (m, 2H), 2.82-2.61 (m, 3H), 2.39-2.18 (m, 2H), 2.20-1.91 (m,6H), 1.78-1.54 (m, 16H), 1.51-1.19 (m, 11H), 1.09-0.82 (m, 17H),0.74-0.62 (m, 1H).

Example 45: Synthesis of(21E,23E,25E,26E,50R,51S,52R,53R,55S,57S,60S,61R,62R,71R)-61,71-dihydroxy-59-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-60-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-62-methoxy-50,51,52,53,63,64-hexamethyl-82,83-dioxa-72-azatricyclohexatriaconta-21,23,25(63),26(64)-tetraene-65,66,67,68,69-pentone(I-96)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of(21E,23E,25E,26E,50R,51S,52R,53R,55S,57S,60S,61R,62R,71R)-61,71-dihydroxy-59-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-60-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-62-methoxy-50,51,52,53,63,64-hexamethyl-82,83-dioxa-72-azatricyclohexatriaconta-21,23,25(63),26(64)-tetraene-65,66,67,68,69-pentone(I-96)

To a solution of rapamycin (0.2 g, 0.22 mmol) and4-methylbenzenesulfonic acid (0.19 g, 1.09 mmol) in THF (6 mL) was added2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol(1.10 g, 2.19 mmol) at 0° C. and the reaction was stirred at 20° C. for2 h then poured into sat. NaHCO₃(30 mL) and extracted with EtOAc (40mL×3). The combined organic layers were washed with water (40 mL), brine(40 mL), dried over anhydrous Na₂SO₄, filtered and concentrated. Theresidue was purified by reverse phase chromatography (C18, CH₃CN:H₂O=51:49) to obtain(21E,23E,25E,26E,50R,51S,52R,53R,55S,57S,60S,61R,62R,71R)-61,71-dihydroxy-59-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-60-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-62-methoxy-50,51,52,53,63,64-hexamethyl-82,83-dioxa-72-azatricyclohexatriaconta-21,23,25(63),26(64)-tetraene-65,66,67,68,69-pentone(I-96: 61 mg, 20% yield) as a colorless oil. ESI-MS (EI⁺, m/z): 1408.0[M+Na]⁺. ¹H NMR (500 MHz, CDCl3) δ 6.41-5.84 (m, 4H), 5.61-5.08 (m, 4H),4.79 (s, 1H), 4.32-4.05 (m, 1H), 3.89-3.46 (m, 44H), 3.46-3.16 (m, 12H),2.98-2.50 (m, 7H), 2.32 (s, 2H), 2.15-1.89 (m, 4H), 1.82-1.68 (m, 8H),1.56-1.16 (m, 14H), 1.12-0.82 (m, 18H), 0.74-0.56 (m, 1H).

Example 46: Synthesis of(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-(2,2,2-trifluoroethoxy)ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone(I-77) and(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,42S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-(2,2,2-trifluoroethoxy)ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentoneand(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,42S,43R,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-(2,2,2-trifluoroethoxy)ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone

Synthetic Scheme:

Procedures and Characterization:

Step 1: Synthesis of(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-(2,2,2-trifluoroethoxy)ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone(I-77)

A solution of rapamycin (0.3 g, 0.313 mmol) and2-(2,2,2-trifluoroethoxy)ethanol (0.9 g, 6.26 mmol) in THF (9 mL) wascooled to 0° C. and p-TsOH (0.27 g, 1.57 mmol) was added. The resultingmixture was stirred at 35° C. for 5h under N₂ then poured into ice coldNaHCO₃ and extracted with EtOAc (40 mL×3). The combined organic layerswere washed with water (30 mL), brine (30 mL), dried, filtered, andconcentrated. The residue was purified via reverse phase chromatography(C18, CH₃CN:H₂O=57:43) to provide(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-(2,2,2-trifluoroethoxy)ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone(I-77: 0.063 g, 19% yield) as a white solid. ESI-MS (EI⁺, m/z): 1092.3[M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ 6.55-5.88 (m, 4H), 5.72-5.03 (m, 4H),4.78 (s, 1H), 4.63-4.34 (m, 1H), 4.32-4.09 (m, 1H), 4.00-2.81 (m, 21H),2.77-2.43 (m, 3H), 2.41-2.17 (m, 2H), 2.18-1.93 (m, 3H), 1.93-1.54 (m,18H), 1.54-1.14 (m, 10H), 1.13-0.79 (m, 16H), 0.78-0.63 (m, 1H).

Step 2: Synthesis of(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,42S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-(2,2,2-trifluoroethoxy)ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone(I-71) and(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,42S,43R,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-(2,2,2-trifluoroethoxy)ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone(I-70)

100 mg of(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-(2,2,2-trifluoroethoxy)ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentonewas purified via prep chiral HPLC and the resulting epimers purified viasilica gel chromatography (hexane:DCM:EtOAc:MeOH=3:3:1:0.3) to provide(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,42S,43S,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-(2,2,2-trifluoroethoxy)ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone(I-71: 16 mg, 16% yield) and(21E,23E,25E,26E,33R,34S,35R,36R,38S,40S,42S,43R,44R,45R,54R)-44,54-dihydroxy-43-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-45-methoxy-33,34,35,36,46,47-hexamethyl-42-[2-(2,2,2-trifluoroethoxy)ethoxy]-65,66-dioxa-56-azatricyclohexatriaconta-21,23,25(46),26(47)-tetraene-48,49,50,51,52-pentone(I-70: 8 mg, 8% yield) as a white solid.

Chiral Separation Method:

Column: CHIRALPAK IC

Column size: 2.5 cm I.D.×25 cm L

Solution concentration: 2.0 mg/ml

Injection: 7 ml

Mobile phase: Hexane/EtOH=70/30(V/V)

Flow rate: 40 ml/min

Wave length: UV 254 nm

Temperature: 35° C.

I-71: ESI-MS (EI⁺, m/z): 1092.4 [M+Na]⁺. ¹H NMR (400 MHz, CDCl3) δ6.41-6.20 (m, 2H), 6.13 (dd, J=15.1, 9.7 Hz, 1H), 5.93 (dd, J=22.9, 10.3Hz, 1H), 5.58-5.45 (m, 1H), 5.41 (d, J=9.8 Hz, 1H), 5.27 (d, J=5.2 Hz,1H), 5.19-5.03 (m, 1H), 4.78 (s, 1H), 4.19 (dd, J=13.9, 5.9 Hz, 1H),3.95-3.63 (m, 10H), 3.63-3.53 (m, 2H), 3.52-3.25 (m, 11H), 3.24-3.01 (m,3H), 2.72 (dd, J=16.8, 5.8 Hz, 2H), 2.58 (dd, J=16.8, 6.3 Hz, 1H), 2.31(t, J=23.5 Hz, 2H), 2.15-1.40 (m, 18H), 1.27 (ddd, J=32.5, 16.2, 6.3 Hz,8H), 1.15-0.81 (m, 18H), 0.70 (dt, J=17.8, 9.0 Hz, 1H).

I-70: ESI-MS (EI⁺, m/z): 1092.4 [M+Na]⁺. ¹H NMR (400 MHz, CDCl3) δ6.43-5.90 (m, 4H), 5.56-5.08 (m, 5H), 4.33-3.99 (m, 3H), 3.95-3.63 (m,8H), 3.62-3.02 (m, 18H), 2.89-1.97 (m, 12H), 1.76 (dd, J=31.4, 24.8 Hz,8H), 1.40 (ddd, J=39.2, 29.5, 12.0 Hz, 9H), 1.14-0.79 (m, 18H),0.76-0.61 (m, 1H).

Example 47: Synthesis of4-[[(23E,25E,27E,28E,35R,36S,37R,38R,40S,42S,45S,46R,47R,57R)-46,57-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-35,36,37,38,48,49-hexamethyl-50,51,52,53,54-pentaoxo-69,70-dioxa-58-azatricyclohexatriaconta-23,25,27(48),28(49)-tetraen-44-yl]oxy]-N,N-dimethyl-butanamide(I-69) and4-[[(23E,25E,27E,28E,35R,36S,37R,38R,40S,42S,44R,45S,46R,47R,57R)-46,57-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-35,36,37,38,48,49-hexamethyl-50,51,52,53,54-pentaoxo-69,70-dioxa-58-azatricyclohexatriaconta-23,25,27(48),28(49)-tetraen-44-yl]oxy]-N,N-dimethyl-butanamide(I-61) and4-[[(23E,25E,27E,28E,35R,36S,37R,38R,40S,42S,44S,45S,46R,47R,57R)-46,57-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-35,36,37,38,48,49-hexamethyl-50,51,52,53,54-pentaoxo-69,70-dioxa-58-azatricyclohexatriaconta-23,25,27(48),28(49)-tetraen-44-yl]oxy]-N,N-dimethyl-butanamide(I-62)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of4-hydroxy-N,N-dimethylbutanamide

A mixture of tetrahydrofuran-2-one (5 g, 58.1 mmol) andN-methylmethanamine (43.64 g, 290.4 mmol, 90 mL) was stirred at 10° C.for 18 h, the solvent was removed, and then lyophilized to afford4-hydroxy-N,N-dimethyl-butanamide (6.9 g, 91% yield) as a colorless oil.ESI-MS (EI⁺, m/z): 132.3 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 3.67 (t,J=5.8 Hz, 2H), 3.04 (s, 3H), 2.96 (s, 3H), 2.49 (t, J=6.9 Hz, 2H),1.94-1.84 (m, 2H).

Step 2: Synthesis of4-[[(23E,25E,27E,28E,35R,36S,37R,38R,4S,42S,45S,46R,47R,57R)-46,57-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-35,36,37,38,48,49-hexamethyl-50,51,52,53,54-pentaoxo-69,70-dioxa-58-azatricyclohexatriaconta-23,25,27(48),28(49)-tetraen-44-yl]oxy]-N,N-dimethyl-butanamide(I-69)

To a solution of everolimus (0.3 g, 0.313 mmol) in THF (9 mL) at 0° C.under N₂ was added p-toluenesulfonic acid (0.27 g, 1.57 mmol) and4-hydroxy-N,N-dimethyl-butanamide (0.82 g, 6.26 mmol). The mixture waswarmed to 35° C. and stirred for 18 h then poured into ice cold NaHCO₃and extracted with EtOAc (20 mL×3). The combined organic layers werewashed with water (30 mL), brine (30 mL), dried over anhydrous Na₂SO₄,filtered, and concentrated. The residue was purified by reverse phasechromatography (C18, CH₃CN:H₂O=55:45) to provide4-[[(23E,25E,27E,28E,35R,36S,37R,38R,40S,42S,45S,46R,47R,57R)-46,57-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-35,36,37,38,48,49-hexamethyl-50,51,52,53,54-pentaoxo-69,70-dioxa-58-azatricyclohexatriaconta-23,25,27(48),28(49)-tetraen-44-yl]oxy]-N,N-dimethyl-butanamide(I-69: 0.05 g, 15% yield) as a white solid. ESI-MS (EI⁺, m/z): 1079.4[M+Na]⁺. ¹H NMR (400 MHz, CDCl3) δ 6.52-5.78 (m, 4H), 5.67-5.05 (m, 4H),4.75 (s, 1H), 4.48-4.09 (m, 2H), 4.04-3.51 (m, 7H), 3.52-3.12 (m, 12H),3.12-2.88 (m, 7H), 2.82-2.27 (m, 6H), 2.22-1.53 (m, 23H), 1.54-1.13 (m,10H), 1.12-0.80 (m, 15H), 0.79-0.61 (m, 1H).

Step 3: Synthesis of4-[[(23E,25E,27E,28E,35R,36S,37R,38R,40S,42S,44S,45S,46R,47R,57R)-46,57-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-35,36,37,38,48,49-hexamethyl-50,51,52,53,54-pentaoxo-69,70-dioxa-58-azatricyclohexatriaconta-23,25,27(48),28(49)-tetraen-44-yl]oxy]-N,N-dimethyl-butanamide(I-62) and4-[[(23E,25E,27E,28E,35R,36S,37R,38R,40S,42S,44R,45S,46R,47R,57R)-46,57-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-35,36,37,38,48,49-hexamethyl-50,51,52,53,54-pentaoxo-69,70-dioxa-58-azatricyclohexatriaconta-23,25,27(48),28(49)-tetraen-44-yl]oxy]-N,N-dimethyl-butanamide(I-61)

120 mg of4-[[(23E,25E,27E,28E,35R,36S,37R,38R,40S,42S,45S,46R,47R,57R)-46,57-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-35,36,37,38,48,49-hexamethyl-50,51,52,53,54-pentaoxo-69,70-dioxa-58-azatricyclohexatriaconta-23,25,27(48),28(49)-tetraen-44-yl]oxy]-N,N-dimethyl-butanamidewas purified via prep chiral HPLC to provide4-[[(23E,25E,27E,28E,35R,36S,37R,38R,40S,42S,44S,45S,46R,47R,57R)-46,57-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-35,36,37,38,48,49-hexamethyl-50,51,52,53,54-pentaoxo-69,70-dioxa-58-azatricyclohexatriaconta-23,25,27(48),28(49)-tetraen-44-yl]oxy]-N,N-dimethyl-butanamide(I-62: 34.3 mg, 29% yield) and4-[[(23E,25E,27E,28E,35R,36S,37R,38R,40S,42S,44R,45S,46R,47R,57R)-46,57-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-35,36,37,38,48,49-hexamethyl-50,51,52,53,54-pentaoxo-69,70-dioxa-58-azatricyclohexatriaconta-23,25,27(48),28(49)-tetraen-44-yl]oxy]-N,N-dimethyl-butanamide(I-61: 24.2 mg, 20% yield), both as white solids.

Chiral Separation Method:

Column: CHIRALPAK IC

Column size: 5.0 cm I.D.×25 cm L

Solution concentration: 1 mg/ml

Injection: 5 ml

Mobile phase: EtOH=100%

Flow rate: 50 ml/min

Wave length: UV 254 nm

Temperature: 35° C.

I-62: ESI-MS (EI⁺, m/z): 1079.4 [M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ 6.30(tt, J=34.4, 17.0 Hz, 2H), 6.13 (dd, J=14.9, 10.0 Hz, 1H), 5.91 (dd,J=28.8, 10.6 Hz, 1H), 5.51 (dd, J=15.0, 8.9 Hz, 1H), 5.45-5.37 (m, 1H),5.27 (d, J=5.4 Hz, 1H), 5.17 (d, J=4.4 Hz, 1H), 4.75 (s, 1H), 4.19 (d,J=4.9 Hz, 1H), 3.88 (s, 1H), 3.82-3.64 (m, 5H), 3.62-3.52 (m, 2H),3.47-3.25 (m, 11H), 3.24-3.14 (m, 2H), 3.10 (d, J=7.0 Hz, 1H), 3.01 (s,3H), 2.94 (s, 3H), 2.71 (dd, J=16.7, 5.7 Hz, 2H), 2.55 (dd, J=16.8, 6.6Hz, 1H), 2.44-2.25 (m, 4H), 2.14-1.63 (m, 17H), 1.33 (ddd, J=40.8, 27.4,12.3 Hz, 11H), 1.14-0.83 (m, 18H), 0.71 (dd, J=23.8, 11.9 Hz, 1H).

I-61: ESI-MS (EI⁺, m/z): 1079.3 [M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ6.43-5.88 (m, 4H), 5.65-5.08 (m, 5H), 4.33-4.08 (m, 2H), 3.94-3.52 (m,6H), 3.49-3.31 (m, 8H), 3.30-3.12 (m, 8H), 3.09-2.81 (m, 8H), 2.75-2.26(m, 6H), 2.10 (d, J=63.9 Hz, 3H), 1.88-1.65 (m, 14H), 1.35 (dt, J=49.7,11.3 Hz, 9H), 1.18-0.81 (m, 18H), 0.77-0.60 (m, 1H).

Example 48: Synthesis of4-[[(23E,25E,27E,28E,35R,36S,37R,38R,40S,42S,45S,46R,47R,57R)-46,57-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-35,36,37,38,48,49-hexamethyl-50,51,52,53,54-pentaoxo-69,70-dioxa-58-azatricyclohexatriaconta-23,25,27(48),28(49)-tetraen-44-yl]oxy]-N,N-dimethyl-butanamide(I-68) and4-[[(22E,24E,26E,27E,34R,35S,36R,37R,39S,41S,43R,44S,45R,46R,56R)-45,56-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-49,50,51,52,53-pentaoxo-68,69-dioxa-58-azatricyclohexatriaconta-22,24,26(47),27(48)-tetraen-43-yl]oxy]-N-methyl-butanamide(I-59) and4-[[(22E,24E,26E,27E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,56R)-45,56-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-49,50,51,52,53-pentaoxo-68,69-dioxa-58-azatricyclohexatriaconta-22,24,26(47),27(48)-tetraen-43-yl]oxy]-N-methyl-butanamide(I-60)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of4-hydroxy-N,N-dimethylbutanamide

To a solution of methylamine (5.41 g, 174.24 mmol) in water (30 mL) wasadded tetrahydrofuran-2-one (5 g, 58.08 mmol) dropwise at 0° C. Themixture was stirred at 5° C. for 2 h then concentrated and lyophilizedto afford 4-hydroxy-N-methyl-butanamide (6.5 g, 95.5% yield) as a thickliquid. ESI-MS (EI⁺, m/z): 118.3 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 6.02(s, 1H), 3.69 (t, J=5.7 Hz, 2H), 2.81 (d, J=4.8 Hz, 3H), 2.36 (t, J=6.8Hz, 2H), 1.88 (dt, J=12.2, 6.1 Hz, 2H).

Step 2: Synthesis of4-[[(22E,24E,26E,27E,34R,35S,36R,37R,39S,41S,44S,45R,46R,56R)-45,56-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-49,50,51,52,53-pentaoxo-68,69-dioxa-58-azatricyclohexatriaconta-22,24,26(47),27(48)-tetraen-43-yl]oxy]-N-methyl-butanamide(I-68)

A solution of everolimus (1 g, 1.04 mmol) and4-hydroxy-N-methyl-butanamide (2.45 g, 20.87 mmol) in THF (30 mL) wascooled to 0° C. under N₂ and p-toluenesulfonic acid (0.9 g, 5.22 mmol)added. The reaction was warmed to 35° C. and stirred for 18 h thenpoured into sat. NaHCO₃ (150 mL) and extracted with EtOAc (100 mL×3).The combined organic layers were washed with water (80 mL), brine (80mL), dried over anhydrous Na₂SO₄, filtered and concentrated. The residuewas purified by reverse phase chromatography (C18, 80 g,CH₃CN:H₂O=37:33) to provide4-[[(22E,24E,26E,27E,34R,35S,36R,37R,39S,41S,44S,45R,46R,56R)-45,56-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-49,50,51,52,53-pentaoxo-68,69-dioxa-58-azatricyclohexatriaconta-22,24,26(47),27(48)-tetraen-43-yl]oxy]-N-methyl-butanamide(I-68: 0.14 g, 13% yield) as a white solid. ESI-MS (EI⁺, m/z): 1065.3[M+Na]⁺. ¹H NMR (400 MHz, CDCl3) δ 6.50-5.85 (m, 4H), 5.78-4.96 (m, 5H),4.78 (s, 1H), 4.33-4.03 (m, 2H), 3.98-3.64 (m, 5H), 3.63-3.49 (m, 2H),3.49-2.90 (m, 13H), 2.90-2.48 (m, 6H), 2.41-1.94 (m, 7H), 1.93-1.54 (m,18H), 1.53-1.11 (m, 10H), 1.11-0.80 (m, 16H), 0.78-0.54 (m, 1H).

Step 3: Synthesis of4-[[(22E,24E,26E,27E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,56R)-45,56-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-49,50,51,52,53-pentaoxo-68,69-dioxa-58-azatricyclohexatriaconta-22,24,26(47),27(48)-tetraen-43-yl]oxy]-N-methyl-butanamide(I-60) and4-[[(22E,24E,26E,27E,34R,35S,36R,37R,39S,41S,43R,44S,45R,46R,56R)-45,56-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-49,50,51,52,53-pentaoxo-68,69-dioxa-58-azatricyclohexatriaconta-22,24,26(47),27(48)-tetraen-43-yl]oxy]-N-methyl-butanamide(I-59)

130 mg of4-[[(22E,24E,26E,27E,34R,35S,36R,37R,39S,41S,44S,45R,46R,56R)-45,56-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-49,50,51,52,53-pentaoxo-68,69-dioxa-58-azatricyclohexatriaconta-22,24,26(47),27(48)-tetraen-43-yl]oxy]-N-methyl-butanamidewas purified via prep chiral HPLC and the resulting epimers purified viasilica gel chromatography (hexane:DCM:EtOAc:MeOH=3:3:1:0.8) to provide4-[[(22E,24E,26E,27E,34R,35S,36R,37R,39S,41S,43S,44S,45R,46R,56R)-45,56-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-49,50,51,52,53-pentaoxo-68,69-dioxa-58-azatricyclohexatriaconta-22,24,26(47),27(48)-tetraen-43-yl]oxy]-N-methyl-butanamide(I-60:mg, 19% yield) and4-[[(22E,24E,26E,27E,34R,35S,36R,37R,39S,41S,43R,44S,45R,46R,56R)-45,56-dihydroxy-44-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-49,50,51,52,53-pentaoxo-68,69-dioxa-58-azatricyclohexatriaconta-22,24,26(47),27(48)-tetraen-43-yl]oxy]-N-methyl-butanamide(I-59: 36 mg, 27% yield), both as white solids.

Chiral Analysis Method:

Column: CHIRALPAK IC-3(IC30CE-NJ008)

Column size: 0.46 cm I.D.×25 cm L

Injection: 50.0 ul

Mobile phase: Hexane/EtOH=50/50(V/V)

Flow rate: 1.0 ml/min

Wave length: UV 254 nm

Temperature: 35° C.

HPLC equipment: Shimadzu LC-20AT CP-HPLC-06

I-60: ESI-MS (EI⁺, m/z): 1065.4 [M+Na]⁺. ¹H NMR (400 MHz, CDCl3) δ 6.32(ddd, J=31.4, 14.8, 10.2 Hz, 2H), 6.13 (dd, J=15.1, 9.9 Hz, 1H),6.00-5.85 (m, 1H), 5.69 (s, 1H), 5.51 (dd, J=14.9, 9.0 Hz, 1H), 5.41 (d,J=9.9 Hz, 1H), 5.27 (d, J=5.7 Hz, 1H), 5.13 (dt, J=48.5, 24.3 Hz, 1H),4.77 (s, 1H), 4.18 (d, J=5.7 Hz, 1H), 3.92-3.63 (m, 6H), 3.61-3.50 (m,2H), 3.46-3.25 (m, 10H), 3.22-3.00 (m, 3H), 2.79 (dd, J=4.8, 2.2 Hz,3H), 2.72 (dd, J=16.9, 5.5 Hz, 2H), 2.55 (dd, J=16.8, 6.5 Hz, 1H),2.38-2.14 (m, 4H), 2.12-1.91 (m, 4H), 1.89-1.62 (m, 15H), 1.52-1.11 (m,13H), 1.10-1.01 (m, 6H), 1.00-0.81 (m, 9H), 0.71 (dd, J=23.6, 12.1 Hz,1H).

I-59: ESI-MS (EI⁺, m/z): 1065.4 [M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ6.37-5.81 (m, 4H), 5.66 (d, J=15.6 Hz, 1H), 5.60-4.89 (m, 4H), 4.16(ddd, J=31.9, 15.6, 5.8 Hz, 2H), 3.93-3.42 (m, 8H), 3.38-2.95 (m, 12H),2.89-2.39 (m, 7H), 2.37-1.89 (m, 9H), 1.83-1.60 (m, 13H), 1.45-1.05 (m,11H), 1.03-0.73 (m, 18H), 0.62 (dd, J=23.9, 12.1 Hz, 1H).

Example 49: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-28)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-28)

To a solution of rapamycin (0.5 g, 0.547 mmol) in THF (10 mL) at 20° C.under N₂ was added 4-methylbenzenesulfonic acid hydrate (0.52 g, 2.73mmol) slowly and 2-[2-(2-hydroxyethoxy)ethoxy]ethanol (1.72 g, 11.49mmol, 3 mL). The resulting solution was stirred for 2h then poured intosat. NaHCO₃(80 mL) and extracted with EtOAc (60 mL×3). The combinedorganic layers were washed with water (60 mL), brine (60 mL), dried overanhydrous Na₂SO₄, filtered and concentrated. The residue was purified byreverse phase chromatography (C-18, CH₃CN:H₂O=75:25) to provide(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-28: 125 mg, 22% yield) as a white solid. ESI-MS (EI⁺, m/z): 1054.5[M+Na]⁺. ¹H NMR (500 MHz, CDCl3) δ 6.44-5.87 (m, 4H), 5.52-5.12 (m, 4H),4.90 (s, 1H), 4.34-4.11 (m, 1H), 4.05-3.83 (m, 1H), 3.80-3.53 (m, 13H),3.50-3.22 (m, 12H), 3.01-2.49 (m, 6H), 2.39-1.87 (m, 6H), 1.82-1.68 (m,8H), 1.44-1.15 (m, 13H), 1.11-0.85 (m, 18H), 0.71-0.57 (m, 1H).

Example 50: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43R,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-30)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43R,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-30)

7.0 g of the epimeric mixture (Example 49; I-28) was purified via prepchiral HPLC to obtain(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43R,44S,45R,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-30: 1.113 g, 16% yield) as a white solid. ESI-MS (EI⁺, m/z): 1054.4[M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ 6.41-5.92 (m, 4H), 5.63 (ddd, J=23.1,15.1, 8.2 Hz, 1H), 5.47 (dd, J=29.9, 10.3 Hz, 1H), 5.30-5.00 (m, 2H),4.33-4.12 (m, 2H), 3.97 (dd, J=18.9, 6.6 Hz, 1H), 3.86-3.48 (m, 14H),3.44-3.22 (m, 10H), 2.97-2.88 (m, 1H), 2.82 (s, 1H), 2.74-2.46 (m, 3H),2.30 (d, J=14.3 Hz, 2H), 2.21-1.91 (m, 5H), 1.86-1.57 (m, 11H),1.50-1.22 (m, 12H), 1.16-0.81 (m, 18H), 0.66 (dt, J=23.8, 11.7 Hz, 1H).

Example 51: Synthesis of(22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,45S,46S,47R,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-44-[2-(2-methoxyethoxy)ethoxy]-35,36,37,38,48,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone(I-121)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of(22E,24E,26E,27E,31R,32S,33R,34R,36S,38S,40S,41S,42S,43R,52R)-42,52-dihydroxy-41-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-40,43-dimethoxy-31,32,33,34,44,45-hexamethyl-62,63-dioxa-53-azatricyclohexatriaconta-22,24,26(44),27(45)-tetraene-46,47,48,49,50-pentone

To a solution of everolimus (1 g, 1.04 mmol) in DCM (50 mL) was addedTi(OiPr)₄ (0.89 g, 3.13 mmol) dropwise at rt. The reaction mixtureturned pale yellow. After 30 min, the solution was poured into aseparatory funnel containing a heterogeneous mixture of 1N HCl (50 mL)and EtOAc(50 mL). The organic layer was sequentially washed withsaturated aqueous NaHCO₃ (30 mL), H₂O (50 mL), brine (50 mL), dried overanhydrous Na₂SO₄, filtered and concentrated under vacuum. The residuewas purified by silica gel chromatography (hexane:acetone=2:1) to obtain(22E,24E,26E,27E,31R,32S,33R,34R,36S,38S,40S,41S,42S,43R,52R)-42,52-dihydroxy-41-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-40,43-dimethoxy-31,32,33,34,44,45-hexamethyl-62,63-dioxa-53-azatricyclohexatriaconta-22,24,26(44),27(45)-tetraene-46,47,48,49,50-pentone(380 mg, 38% yield) as a light yellow solid. ¹H NMR (400 MHz, DMSO-d6) δ6.44 (dt, J=13.9, 10.3 Hz, 2H), 6.33-6.06 (m, 3H), 5.54-4.85 (m, 5H),4.48 (t, J=5.3 Hz, 1H), 4.10-3.91 (m, 2H), 3.89-3.79 (m, 1H), 3.62 (d,J=11.5 Hz, 1H), 3.54-3.41 (m, 5H), 3.38-3.28 (m, 8H), 3.19 (dt, J=11.9,7.6 Hz, 4H), 3.10-2.92 (m, 6H), 2.71 (t, J=14.8 Hz, 1H), 2.43-1.78 (m,6H), 1.75-1.44 (m, 10H), 1.38-0.90 (m, 14H), 0.89-0.67 (m, 13H),0.66-0.56 (m, 1H).

Step 2: Synthesis of(22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,45S,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-44-[2-(2-methoxyethoxy)ethoxy]-35,36,37,38,48,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone(I-121)

To a solution of 28-epi-everolimus (0.2 g, 0.208 mmol) and2-(2-methoxyethoxy)ethanol (0.99 mL, 8.35 mmol) in sulfolane (5 mL) wasadded HND-8 (35 mg) at 50° C. under N₂. The resulting solution wasstirred at 50° C. for 4 h, filtered and diluted with water (30 mL) andEtOAc (30 mL). The organic layer was washed with water (10 mL×3), brine(20 mL), dried over anhydrous Na₂SO₄, filtered and concentrated. Theresidue was purified via reverse-phase chromatography (C18, CH₃CN:H₂O=6.5: 3.5) to provide(22E,24E,26E,27E,35R,36S,37R,38R,40S,42S,45S,56R)-46,56-dihydroxy-45-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxy-cyclohexyl]-1-methyl-ethyl]-47-methoxy-44-[2-(2-methoxyethoxy)ethoxy]-35,36,37,38,48,49-hexamethyl-66,67-dioxa-57-azatricyclohexatriaconta-22,24,26(48),27(49)-tetraene-50,51,52,53,54-pentone(I-121: 60 mg, 27% yield) as a white solid. ESI-MS (EI⁺, m/z): 1068.1[M+Na]⁺. ¹H NMR (500 MHz, DMSO-d₆) δ 6.57-6.35 (m, 2H), 6.31-5.88 (m,3H), 5.61-5.38 (m, 1H), 5.28-4.82 (m, 4H), 4.47 (t, J=5.3 Hz, 1H), 3.98(dd, J=40.4, 6.9 Hz, 2H), 3.88-3.73 (m, 2H), 3.59-3.40 (m, 12H), 3.30(dd, J=12.1, 8.2 Hz, 4H), 3.26-3.14 (m, 8H), 3.08-2.92 (m, 3H),2.85-2.62 (m, 2H), 2.43-2.22 (m, 2H), 2.19-1.82 (m, 6H), 1.79-1.45 (m,9H), 1.32 (dd, J=58.1, 21.2 Hz, 5H), 1.19-0.92 (m, 10H), 0.90-0.57 (m,15H).

Example 52: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45S,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-127) and(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45S,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-128) and(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43R,44S,45S,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-129)

Synthetic Scheme:

Procedures and Characterization: Step 1: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45S,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-127)

To a solution of 28-epi-rapamycin (0.2 g, 0.22 mmol; see Example 52) and2-[2-(2-hydroxyethoxy)ethoxy]ethanol (0.436 mL, 3.28 mmol) in sulfolane(5 mL) was added HND-8 (30 mg) and the mixture stirred at 50° C. for 5h. Upon cooling, the reaction was diluted with EtOAc (50 mL), filtered,washed with water (50 mL×3) and brine(50 mL), dried over anhydrousNa₂SO₄, filtered and concentrated. The residue was purified by reversephase chromatography (C18, CH₃CN:H₂O from 0 to 70% yield) to provide(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45S,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-127: 60 mg, 26% yield) as a light yellow solid. ESI-MS (EI⁺, m/z):1054.4 [M+Na]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 6.61-6.32 (m, 2H), 6.30-5.98(m, 3H), 5.65-5.38 (m, 1H), 5.28-4.89 (m, 4H), 4.68-4.53 (m, 2H),4.11-3.91 (m, 2H), 3.89-3.71 (m, 2H), 3.54-3.44 (m, 9H), 3.44-3.40 (m,3H), 3.31-3.28 (m, 4H), 3.27-3.11 (m, 6H), 2.86-2.66 (m, 3H), 2.20-1.82(m, 7H), 1.80-1.58 (m, 13H), 1.42-1.06 (m, 9H), 1.05-0.68 (m, 18H), 0.56(dd, J=23.8, 12.0 Hz, 1H).

Step 2: Synthesis of(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45S,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-128) and(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43R,44S,45S,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-129)

130 mg of(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,44S,45S,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentonewas purified via prep chiral HPLC and the resulting epimers purified viasilica gel chromatography (hexane:DCM:EtOAc:MeOH=3:3:1:0 to 3:3:1:0.8)to provide(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43S,44S,45S,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-128: 28 mg, 21.5% yield) and(21E,23E,25E,26E,34R,35S,36R,37R,39S,41S,43R,44S,45S,46R,55R)-45,55-dihydroxy-43-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-44-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxy-cyclohexyl]-1-methyl-ethyl]-46-methoxy-34,35,36,37,47,48-hexamethyl-66,67-dioxa-56-azatricyclohexatriaconta-21,23,25(47),26(48)-tetraene-49,50,51,52,53-pentone(I-129: 22 mg, 16.9% yield), both as white solids.

Chiral Separation Method:

Column: CHIRALPAK IC(IC00CE-WF029)

Column size: 0.46 cm I.D.×25 cm L

Injection: 10.0 ul

Mobile phase: Hexane/EtOH=60/40(V/V)

Flow rate: 1.0 ml/min

Wave length: UV 254 nm

Temperature: 35° C.

HPLC equipment: Shimadzu LC-20AT CP-HPLC-09

I-128: ESI-MS (EI⁺, m/z): 1054.2 [M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ6.56-6.22 (m, 2H), 6.14 (dd, J=15.0, 10.3 Hz, 1H), 6.00 (dd, J=17.9,11.0 Hz, 1H), 5.61-5.46 (m, 1H), 5.46-5.32 (m, 1H), 5.29-5.12 (m, 2H),4.70 (s, 1H), 4.15-4.04 (m, 1H), 3.99-3.88 (m, 1H), 3.86-3.52 (m, 13H),3.51-3.25 (m, 11H), 3.01-2.60 (m, 5H), 2.52 (dd, J=16.7, 7.1 Hz, 2H),2.39-1.87 (m, 7H), 1.74 (dt, J=13.3, 8.7 Hz, 8H), 1.63-1.16 (m, 13H),1.14-0.78 (m, 18H), 0.67 (dd, J=23.8, 12.0 Hz, 1H).

I-129: ESI-MS (EI⁺, m/z): 1054.1 [M+Na]⁺. ¹H NMR (500 MHz, CDCl₃) δ6.46-5.89 (m, 4H), 5.60-4.93 (m, 5H), 4.15 (dd, J=50.5, 20.8 Hz, 2H),3.97-3.15 (m, 25H), 3.06-2.43 (m, 8H), 2.39-1.69 (m, 16H), 1.54-1.19 (m,10H), 1.13-0.79 (m, 18H), 0.66 (dd, J=23.6, 11.7 Hz, 1H).

Example 53: AlphaLISA Ultra pS6K1 Assay

Assay Protocol

-   -   1. Seed MCF-7 cells in Corning 3701 plate and incubate for 20-24        hour. 12,000-16,000 cells will be seeded in 36 μL medium per        well.    -   2. Change the culture medium with fresh medium and incubate for        another 2 hours.    -   3. Add 12 μL (4×) compounds into the cell plate by HAMILTON.        Final DMSO concentration is 0.5%. Incubate for 90 minutes.    -   4. Aspirate 38 μL by HAMILTON, 10 μL rest per well.    -   5. Add 10 μL 2× lysis buffer using HAMILTON; total volume in        wells is 20 μL. Allow cells to shake for 30 min. Cover plate by        plastic foil and store plate at −80° C. up to analysis.    -   6. Thaw cell lysate at R^(†) and transfer 10 μL lysate to assay        plate (Optiplate-384).    -   7. Add 5 μL acceptor beads into assay plate and incubation for 2        hours    -   8. Add 5 μL donor beads and incubation for 2 hours    -   9. Count the plate by EnSpire Multimode Plate Reader

TABLE 2 Key Reagents/Supplies Reagents/materials Vendor Cat. No. Lot.No. MCF-7 ATCC HTB-22 5105360 DMEM Invitrogen 12430-054 1677193 FBSInvitrogen 10099-141 1660516 0.25% Trypsin-EDTA Invitrogen 25200-0721638603 384 well plate, tissue culture Corning CLS3701 29214010 treatedCorning 384 well storage Corning CLS3656 29514036 plates Torin1 SelleckS2827 01 Rapamycin SELLECK S1039 08 OptiPlate-384, White OpaquePerkinElmer 6007299 8210-14501 384-well MicroPlate AlphaLISA SureFireUltra PerkinElmer ALSU-PP70- U0381 p-p70 S6 Kinase (Thr389) A10K AssayKit

Example 54: AlphaLISA Ultra pAKT Assay

Assay Protocol:

-   -   1. MCF-7 cells in Corning 3701 plate and incubate for 20-24        hour. 16,000-20,000 cells will be seeded in 36 μL medium per        well.    -   2. Change the culture medium with fresh medium and incubate for        another 90 minutes.    -   3. Add 12 μL (4×) compounds into the cell plate by HAMILTON.        Final DMSO concentration is 0.5%. Incubate for 2 hours.    -   4. Aspirate 38 μL by HAMILTON, 10 μL rest per well.    -   5. Add 10 μL 2× lysis buffer using HAMILTON; total volume in        wells is 20 μL. Allow cells to shake for 30 min. Cover plate by        plastic foil and store plate at −80° C. up to analysis.    -   6. Thaw cell lysate at R^(†) and transfer 10 ul lysate to assay        plate (Optiplate-384).    -   7. Add 5 μL acceptor beads into assay plate and incubation for 2        hours    -   8. Add 5 μL donor beads and incubation for 2 hours    -   9. Count the plate by EnSpire Multimode Plate Reader

TABLE 3 Key Reagents/Supplies Reagents/materials Vendor Cat. No. Lot.No. MCF-7 ATCC HTB-22 5105360 DMEM Invitrogen 12430-054 1677193 FBSInvitrogen 10099-141 1660516 0.25% Trypsin-EDTA Invitrogen 25200-0721638603 384 well plate, tissue Corning CLS3701 29214010 culture treatedCorning 384 well storage Corning CLS3656 29514036 plates Torin1 SelleckS2827 01 Rapamycin SELLECK S1039 08 OptiPlate-384, White PerkinElmer6007299 8210-14501 Opaque 384-well MicroPlate AlphaLISA SureFire UltraPerkinElmer ALSU-PAKT- U0329 p-Akt 1/2/3 (Ser473) B10K Assay Kits

Example 55: Western Blot Based pS6K1 and pAKT Assay at 24 and 48 HourTimepoints

Assay Protocol

-   -   1. Seed six well plate with 500,000 PC3 cells per well and        incubate for 20-24 hour.    -   2. Add compounds into the cell plate. Incubate for 24 to 48        hours.    -   4. Plate is placed on ice and the media is removed via        aspiration. The wells are washed with 1 mL of 1x PBS and then        fully aspirated.    -   5. 110 μL of 1% Triton Lysis Buffer is added and each well is        scraped vigorously.    -   6. Cell homogenates are transferred to 1.5 mL eppendorf tubes on        ice and spun down at 4° C. for 10 minutes at 10,000 rpm.    -   7. Protein concentration of resulting cell lysates were        quantified utilizing a Bradford assay and the samples run        analyzed via Western blot on 4-12% Bis/Tris gels with 1×MES        buffer.    -   8. The gels were transferred onto membranes at 50V for 100        minutes, blocked with Odyssey Blocking buffer for 30 minutes        then incubated overnight with primary antibody (pS6K1 T389        Rabbit or pAkt S473 Rabbit) overnight at 4° C. on a rotator.    -   9. The membranes were washed 3× with TBS-T with a 5 minute        incubation between each wash then incubated with secondary        antibody (LiCor IRDye 800 Donkey Anti Rabbit) for at least 30        minutes.    -   10. The membranes were washed 3× with TBS-T with a 5 minute        incubation between each wash.    -   11. The gels were then incubated for 5 minutes with PBS at room        temperature then imaged using a Li-Cor.

Results for a representative Western blot are summarized in FIG. 1. PC3cells were treated with rapamycin (0.1 μM and 0.01 μM) or I-29 (1 μM,0.1 μM, 0.01 μM, and 0.001 μM) for 24 and 48 hours. Blots clearlydemonstrate a significant reduction in pS6K1 for both rapamycin and I-29at both 24 and 48 hours, indicating inhibition of the mTORC1 pathway.Importantly, I-29 did not reduce the levels of pAkt at 24 or 48 hours.In contrast, rapamycin exhibited inhibition of S6K1 phosphorylation(S⁴⁷³) at both 24 and 48 hours, indicative of mTORC2 pathway inhibition.

Results for additional representative Western blots, and the compoundsevaluated therein, are summarized in FIG. 2 through FIG. 42. The methodsemployed were substantially similar to those described above. Compoundswere evaluated in PC3 cells, Jurkat cells, wild-type mouse embryoticfibroblast (MEF) cells, tuberous sclerosis 2 (TSC2) negative (TSC−/−)MEF cells, and tuberous sclerosis 2 (TSC2) positive (TSC+/+) MEF cells.Cells were incubated with compounds of the present invention for varioustime periods (e.g., 5 minutes, 15 minutes, 30 minutes, 90 minutes, 24hours, or 48 hours), and evaluated according to known methodologies,such as those herein described.

Table 4 shows the inhibitory activity (IC50) of selected compounds ofthis invention in the pS6K1 and pAKT assays, and their solubility in 100mM phosphate buffer (pH 7.4). The compound numbers correspond to thecompound numbers in Table 1.

Compounds of the present invention that selectively inhibit mTORC1 overmTORC2- and retain selectivity for at least 24 hours—are indicated by“YES” in the “mTORC1 selective @ 24 hrs” column of Table 4. Compoundsthat are not selective at the 24 hrs mark are indicated by “NO” in the“mTORC1 selective @ 24 hrs” column of Table 4. Compounds that partiallyretain selectivity for mTORC1 inhibition over mTORC2 are indicated by“Partial” in the “mTORC1 selective @ 24 hrs” column of Table 4. “N/A”stands for “not assayed.”

Compounds denoted “+” exhibit solubilities less than 30 μM (x<30 μM).Compounds denoted “++” exhibit solubitilies greater then or equal to 30μM and less than 60 μM (30 μM≤x≤60 μM). Compounds denoted “+++” exhibitsolubilities greater than or equal to 60 μM (60 μM≤x).

Compounds denoted “A” exhibited an IC₅₀ lower than 0.1 nM (x<0.1 nM).Compounds denoted “B” exhibited an IC₅₀ greater than or equal to 0.1 nMand less than 1 nM (0.1 nM≤x≤1.0 nM). Compounds denoted “C” exhibited anIC₅₀ greater than or equal to 1.0 nM and less than 10 nM (1.0 nM≤x≤10nM). Compounds denoted “D” exhibited an IC₅₀ greater than or equal to 10nM and les than 100 nM (10 nM≤x≤100 nM). Compounds denoted “E” exhibitedan IC₅₀ greater than or equal to 100 nM (100 nM≤x).

TABLE 4 Assay Data for Exemplary Compounds pS6K1 pAKT in in Aqueous MCF7@ MCF7@ mTORC1 Solubility Compound 90 min: 90 min: selective @ at pH 7.4Number IC₅₀ IC₅₀ 24 hrs (μM) I-1 B E YES ++ I-2 B E YES ++ I-3 B E YES++ I-4 B N/A YES N/A I-5 N/A N/A YES N/A I-6 C E YES N/A I-7 N/A N/A YESN/A I-8 C N/A YES N/A I-9 N/A N/A YES N/A I-10 A E YES N/A I-11 C E YESN/A I-12 N/A N/A YES N/A I-13 C N/A YES N/A I-14 N/A N/A YES N/A I-15 CE YES ++ I-16 B E YES ++ I-17 B E YES ++ I-19 A E YES ++ I-20 B E YES ++I-21 A E YES + I-22 B E YES N/A I-23 A E YES N/A I-24 A E YES + I-25 B EYES N/A I-26 B E YES +++ I-27 B E YES ++ I-30 B E YES +++ I-31 B E YES+++ I-32 B E YES +++ I-33 C E YES +++ I-34 B E NO ++ I-35 B E YES +++I-36 C E YES +++ I-37 C E YES ++ I-38 A E NO ++ I-39 C E YES N/A I-29 AE YES ++ I-57 A N/A YES N/A I-58 B N/A YES N/A I-59 E N/A YES N/A I-60 CN/A YES N/A I-61 C N/A YES N/A I-62 C N/A YES N/A I-63 E N/A YES N/AI-64 C N/A YES N/A I-65 B N/A YES N/A I-66 C N/A YES N/A I-67 B N/A YESN/A I-68 C N/A YES N/A I-69 B N/A YES N/A I-70 C N/A YES N/A I-71 B N/AYES N/A I-72 C N/A YES N/A I-73 C N/A YES N/A I-74 B N/A NO N/A I-75 AN/A YES N/A I-76 C N/A YES N/A I-77 A N/A YES N/A I-78 C N/A YES N/AI-79 C N/A YES N/A I-80 B N/A YES N/A I-81 C N/A YES N/A I-82 B N/A YESN/A I-83 N/A N/A YES N/A I-84 B N/A YES N/A I-85 N/A N/A YES N/A I-86 AN/A YES N/A I-87 A N/A YES N/A I-88 A N/A YES N/A I-89 C N/A YES N/AI-90 N/A YES N/A I-91 A N/A YES N/A I-92 B N/A YES N/A I-95 N/A N/A YESN/A I-96 B N/A YES N/A I-97 C N/A YES N/A I-98 C N/A YES N/A I-99 B N/AYES N/A I-100 C N/A YES N/A I-101 B N/A YES N/A I-102 D N/A YES N/AI-103 B N/A YES N/A I-104 B N/A YES N/A I-105 A N/A YES N/A I-106 B N/AYES N/A I-107 E N/A YES N/A I-108 B N/A YES N/A I-109 C N/A YES N/AI-110 B N/A YES N/A I-111 C N/A YES N/A I-112 B N/A YES N/A I-113 B N/AYES N/A I-114 C N/A YES N/A I-115 B N/A YES N/A I-116 B N/A YES N/AI-117 A N/A YES N/A I-118 C N/A YES N/A I-119 A N/A YES N/A I-120 A N/AYES N/A

Example 56: Pharmacokinetic Properties

The pharmacokinetic properties of compounds of the present inventionwere evaluated in C57Bl/6 mice and compared with rapamycin. Animals werefasted overnight prior to administration of I-29 or rapamycin (1 mg/kgIV, 10 mg/kg PO, or 2 mg/kg IP). Animals were bled at time intervals forupto 48 hours following administration of compounds. Whole blood fromeach mouse was individually collected in polypropylene tubes andimmediately centrifuged. Alloquates of the separated plasma werepromptly prepared for HPCL analysis. The results of the pharmacokineticsstudies are summarized in Table 5. Compound I-29 shows improved oralbioavailability compared to rapamycin, as well as lower clearance,longer half life, increased Cmax, and increased AUC as compared torapamycin.

TABLE 5 Comparison of mouse pharmacokinetic properties of rapamycin andI-29 I-29 Rapamycin 1 mg/kg IV CL (L/hr/Kg) 0.054 0.276 V_(ss) (L/Kg)0.863 1.73 Terminal T_(1/2) (h) 14.7 8.35 AUC_(last) 17591 3616 (h ·ng/mL) AUC_(INF) 18393 3627 (h · ng/mL) MRT_(INF) (h) 15.9 6.27 10 mg/kgT_(max) (h) 0.25 3 PO C_(max) (ng/mL) 3287 52.8 Terminal T_(1/2) (h) 11N/A AUC_(last) 31236 292 (h · ng/mL) AUC_(INF) 31726 N/A (h · ng/mL) F(%) 17.2 0.8 2 mg/kg IP T_(max) (h) 0.25 0.25 C_(max) (ng/mL) 2590 614Terminal T_(1/2) (h) 12.6 4.8 AUC_(last) 16607 2481 (h · ng/mL)AUC_(INF) 16937 2544 (h · ng/mL) F (%) 46 35.1

Example 57: Assessment of Effect of Chronic Treatment with I-29 onGlucose Tolerance and Insulin Sensitivity in Lean C5711/6 Mice

C57Bl/6 mice (n=12; 8 weeks of age) were randomized and baselinemeasurements (weight, fasting glucose, and fasting insulin) weremeasured four (4) days prior to the administration of compounds orvehicle. Animals were then treated with I-29 (10 mg/kg P0), rapamycin(10 mg/kg IP) or vehicle (PO or IP) for 19 days. On day 7 and 14 animalswere weighed and fed glucose and fed insulin levels were evaluated.Animals were fasted overnight on day 14 and on day 15 fasting insulinand an intraperitoneal glucose tolerance test (ipGTT) were evaluated. Onday 19 animals were sacrificed one (1) hour after compound or vehicleadministration. Tissues were harvested for evaluating compound levelsand pharmacodynamics. The time course of the study is summarized in FIG.43.

The results of the ipGTT are summarized in FIG. 44 and FIG. 45. Briefly,chronic rapamycin treatment for 15 days induced glucose intolerance inC57Bl/6 mice, as demonstrated by the elevated glucose levels compared toVh R. In comparison, I-29 did not induce glucose intolerance.

Example 58: Assessment of Effect of I-29, I-117 and Everolimus in theAcute Kidney Disease/Chromic Kidney Disease (AKI/CKD) Mouse Model

C57Bl/6 mice (n=15; male; 10 weeks of age) were randomized. Following 7days of acclimation, IR or sham surgery was performed. Mice were allowedto recover for one (1) day and beginning on day 2 animals receiving theIR surgery were administered vehicle, everolimus (10 mg/kg PO), I-29 (10mg/kg PO), or I-117 (10 mg/kg PO). On day 9 uninephrectomy (Unx) or shamsurgery was performed. On day 29 animals were sacrificed.

Kidney histology was evaluated by PAS, Masson's trichrome staining orSirius red. Results for Sirus red staining are summarized in FIG. 46 andFIG. 47. Briefly, I-29 showed a significant reducing in kidney fibrosisas compared to vehicle.

Kidney tissue mRNA was analysis for inflammatory and fibrotic markers(qPCR for TGFβ, collegen I, collagen III, CCCTC-binding factor (CTCF),fibronectin (FN), and alpha-smooth muscle actin (α-SMA)).

Immunohistochemistry was evaluated for collagen I, collagen IV, u-SMA,4-hydroxynonenal (4-HNE), and F4/80 macrophage.

Plasma was evaluated for compound levels and kidney tissue was evaluatedfor pharmacodynamics.

Results for the expression of fibrosis markers and macrophageinfiltration in the kidneys is summarized in FIG. 48 to FIG. 51.Briefly, I-29 significantly reduced the expression of collagen I,collagen III, and fibronectin mRNA. Further, I-29 significantlydecreased infiltration of macrophage into the kidney.

Example 59: Assessment of Effect on IFN-γ Production in Allogenic MixedLymphocyte Reaction

The effect of rapamycin, everolimus, I-29, and I-117 on IFN-γ productionwas evaluated in an allogenic mixed lymphocyte reaction (E.g.,Eleftheriadis, T. et al., Int. J. Mol. Med., 37(5): 1412-20 (2016)https://doi.org/10.3892/ijmm.2016.2547). IC₅₀ values are summarized inTable 6, below, and FIG. 52. Briefly, I-29 and I-117 did not inhibitIFN-γ production, whereas rapamycin and everolimus significantlyinhibited IFN-γ production.

TABLE 6 IFN-γ Inhibition Rapamycin Everolimus I-29 I-117 IC₅₀s (nM) 2.415.2 >3000 >3000

1.-17. (canceled)
 18. A method of treating an mTORC1-mediated disease,disorder, or condition in a patient in need thereof, comprisingadministering to said patient, a compound of Formula II:

or a pharmaceutically acceptable salt thereof, wherein: R¹ is selectedfrom


19. The method according to claim 18, wherein the mTORC1-mediateddisease, disorder, or condition is selected from: diabetic nephropathy,kidney-related complications of type 1 diabetes and type 2 diabetes,autosomal dominant polycystic kidney disease (ADPKD), autosomalrecessive polycystic kidney disease (ARPKD), kidney diseases associatedwith cyst formation or cystogenesis, focal segmental glomerulosclerosis(FSGS) and other diseases associated with sclerosis of the kidney,laminopathies, age-related macular degeneration (AMD), diabetic macularedema, diabetic retinopathy, glaucoma, age related retinal disease,immune system senescence, respiratory tract infections, urinary tractinfections, heart failure, osteoarthritis, pulmonary arterialhypertension (PAH), and chronic obstructive pulmonary disease (COPD).20. The method according to claim 18, further comprising administeringan additional therapeutic agent in combination with said compound. 21.(canceled)
 22. The method according to claim 18, wherein said compoundof Formula II is selected from:

or a pharmaceutically acceptable salt thereof.
 23. The method accordingto claim 18, wherein the mTORC1-mediated disease, disorder, or conditionis diabetic nephropathy.
 24. The method according to claim 18, whereinthe mTORC1-mediated disease, disorder, or condition is kidney-relatedcomplications of type 1 diabetes and type 2 diabetes.
 25. The methodaccording to claim 18, wherein the mTORC1-mediated disease, disorder, orcondition is autosomal dominant polycystic kidney disease (ADPKD). 26.The method according to claim 18, wherein the mTORC1-mediated disease,disorder, or condition is autosomal recessive polycystic kidney disease(ARPKD).
 27. The method according to claim 18, wherein themTORC1-mediated disease, disorder, or condition is kidney diseasesassociated with cyst formation or cystogenesis.
 28. The method accordingto claim 18, wherein the mTORC1-mediated disease, disorder, or conditionis focal segmental glomerulosclerosis (FSGS) and other diseasesassociated with sclerosis of the kidney.
 29. A method of treatingpolycystic kidney disease (PKD) in a patient in need thereof, comprisingadministering to said patient a compound of Formula II:

or a pharmaceutically acceptable salt thereof, wherein: R¹ is selectedfrom


30. The method according to claim 29, wherein said compound of FormulaII is selected from:

or a pharmaceutically acceptable salt thereof.
 31. The method accordingto claim 29, wherein the PKD is autosomal dominant polycystic kidneydisease (ADPKD).
 32. The method according to claim 29, wherein the PKDis autosomal recessive polycystic kidney disease (ARPKD).
 33. The methodaccording to claim 29, further comprising administering an additionaltherapeutic agent in combination with said compound.
 34. A method oftreating kidney fibrosis in a patient in need thereof, comprisingadministering to said patient a compound of Formula II:

or a pharmaceutically acceptable salt thereof, wherein: R¹ is selectedfrom


35. The method according to claim 34, wherein said compound of FormulaII is selected from:

or a pharmaceutically acceptable salt thereof.
 36. The method accordingto claim 34, wherein the patient suffers from an acute kidney disease(AKD).
 37. The method according to claim 34, wherein the patient suffersfrom a chronic kidney disease (CKD).
 38. The method according to claim34, further comprising administering an additional therapeutic agent incombination with said compound.